High affinity nucleic acid ligands to lectins

ABSTRACT

This invention discloses high-affinity oligonucleotide ligands to lectins, specifically nucleic acid ligands having the ability to bind to the lectins, wheat germ agglutinin, L-selectin, E-selectin and P-selectin. Also disclosed are the methods for obtaining such ligands.

RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 07/714,131, filed Jun. 10, 1991, entitled Nucleic Acid Ligands,now U.S. Pat. No. 5,475,096 which is a Continuation-in Part of U.S.patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitledSystematic Evolution of Ligands by Exponential Enrichment, nowabandoned, and U.S. patent application Ser. No. 07/964,624, filed Oct.21, 1992, entitled Methods of Producing Nucleic Acid Ligands, now U.S.Pat. No. 5,496,938.

FIELD OF THE INVENTION

Described herein are methods for identifying and preparing high-affinitynucleic acid ligands to lectins. Lectins are carbohydrate bindingproteins. The method utilized herein for identifying such nucleic acidligands is called SELEX, an acronym for Systematic Evolution of Ligandsby EXponential enrichment. Specifically disclosed herein arehigh-affinity nucleic acid ligands to wheat germ agglutinin (WGA),L-selectin, E-selectin, and P-selectin.

BACKGROUND OF THE INVENTION

The biological role of lectins (non-enzymatic carbohydrate-bindingproteins of non-immune origin; I. J. Goldstein et al., 1980, Nature285:66) is inextricably linked to that of carbohydrates. One function ofcarbohydrates is the modification of physical characteristics ofglyco-conjugates (ie., solubility, stability, activity, susceptabilityto enzyme or antibody recognition), however, a more interesting andrelevant aspect of carbohydrate biology has emerged in recent years; thecarbohydrate portions of glycoconjugates are information rich molecules(N. Sharon and H. Lis, 1989, Science 246:227-234; K. Drickamer and M.Taylor, 1993, Annu. Rev. Cell Biol. 9:237-264; A. Varki, 1993,Glycobiol. 3:97-130). Within limits, the binding of carbohydrates bylectins is specific (ie., there are lectins that bind only galactose orN-acetylgalactose; other lectins bind mannose; still others bind sialicacid and so on; K. Drickamer and M. Taylor, supra). Specificity ofbinding enables lectins to decode information contained in thecarbohydrate portion of glyco-conjugates and thereby mediate manyimportant biological functions.

Numerous mammalian, plant, microbial and viral lectins have beendescribed (I. Ofek and N. Sharon, 1990, Current Topics in Microbiol. andImmunol. 151:91-113; K. Drickamer and M. Taylor,supra; I. J. Goldsteinand R. D. Poretz, 1986, in The Lectins, pp 33-247; A. Varki, supra).These proteins mediate a diverse array of biological processes whichinclude: trafficking of lysosomal enzymes, clearance of serum proteins,endocytosis, phagocytosis, opsonization, microbial and viral infections,toxin binding, fertilization, immune and inflammatory responses, celladhesion and migration in development and in pathological conditionssuch as metastasis. Roles in symbiosis and host defense have beenproposed for plant lectins but remain controversial. While thefunctional role of some lectins is well understood, that of many othersis understood poorly or not at all.

The diversity and importance of processes mediated by lectins isillustrated by two well documented mammalian lectins, theasialoglycoprotein receptor and the serum mannose binding protein, andby the viral lectin, influenza virus hemagglutinin. The hepaticasialoglycoprotein receptor specifically binds galactose andN-acetylgalactose and thereby mediates the clearance of serumglycoproteins that present terminal N-acetylgalactose or galactoseresidues, exposed by the prior removal of a terminal sialic acid. Thehuman mannose-binding protein (MBP) is a serum protein that bindsterminal mannose, fucose and N-acetylglucosamine residues. Theseterminal residues are common on microbes but not mammalianglyco-conjugates. The binding specificity of MBP constitutes anon-immune mechanism for distinguishing self from non-self and mediateshost defense through opsonization and complement fixation. Influenzavirus hemagglutinin mediates the initial step of infection, attachmentto nasal epithelial cells, by binding sialic acid residues ofcell-surface receptors.

The diversity of lectin mediated functions provides a vast array ofpotential therapeutic targets for lectin antagonists. Both lectins thatbind endogenous carbohydrates and those that bind exogenouscarbohydrates are target candidates. For example, antagonists to themammalian selectins, a family of endogenous carbohydrate bindinglectins, may have therapeutic applications in a variety ofleukocyte-mediated disease states. Inhibition of selectin binding to itsreceptor blocks cellular adhesion and consequently may be useful intreating inflammation, coagulation, transplant rejection, tumormetastasis, rheumatoid arthritis, reperfusion injury, stroke, myocardialinfarction, burns, psoriasis, multiple sclerosis, bacterial sepsis,hypovolaemic and traumatic shock, acute lung injury, and ARDS.

The selectins, E-, P- and L-, are three homologous C-type lectins thatrecognise the tetrasaccharide, sialyl-Lewis^(X) (C. Foxall et al, 1992,J. Cell Biol 117,895-902). Selectins mediate the initial adhesion ofneutrophils and monocytes to activated vascular endothelium at sites ofinflammation (R. S. Cotran et al., 1986, J. Exp. Med 164, 661-; M. A.Jutila et al., 1989, J. Immunol. 143,3318-;J. G. Geng et al, 1990,Nature, 757; U. H. Von Adrian et al., 1994, Am. J. Physiol. Heart Circ.Physiol 263, H1034-H1044). In addition, L-selectin is responsible forthe homing of lymphocytes to peripheral and mesenteric lymph nodes (W.M. Gallatin et al, 1983, Nature 304,30; T. K. Kishimoto et al, 1990,Proc. Natl. Acad. Sci. 87,2244-) and P-selectin mediates the adherenceof platelets to neutrophils and monocytes (S. C. Hsu-Lin et al., 1984,J. Biol. Chem. 259,9121).

Selectin antagonists (antibodies and carbohydrates) have been shown toblock the extravasation of neutrophils at sites of inflammation (P.Piscueta and F. W. Luscinskas, 1994, Am. J. Pathol. 145, 461-469), to beeffacacious in animal models of ischemia/reperfusion (A. S. Weyrich etal., 1993, J. Clin. Invest. 91,2620-2629; R. K. Winn et al., 1993, J.Clin. Invest. 92, 2042-2047), acute lung injury (M. S. Mulligan et al.,1993, J. Immunol. 151, 6410-6417; A. Seekamp it al., 1994, Am. J Pathol.144, 592-598), insulitis/diabetes (X. D. Yang et al., 1993, Proc. Natl.Acad. Sci. 90,10494-10498), meningitis (C. Granet et al., 1994, J. Clin.Invest. 93, 929-936), hemorrhagic shock (R. K. Winn et al., 1994, Am J.Physiol. Heart Circ. Physiol. 267, H2391-H2397) and transplantation. Inaddition, selectin expression has been documented in models of arthritis(F. Jamar et al., 1995, Radiology 194, 843-850), experimental allergicencephalomyelitis (J. M. Dopp et al., 1994, J. Neuroimmunol. 54,129-144), cutaneous inflammation (A. Siber et al., 1994, Lab. Invest.70, 163-170) glomerulonephritis (P. G. Tipping et al., 1994, Kidney Int.46, 79-88), on leukaemic cells and colon carcinomas (R. M. Lafrenie etal., 1994, Eur. J. Cancer A! 30A, 2151-2158) and L-selectin receptorshave been observed in myelinated regions of the central nervous system(K. Huang et al., 1991, J. Clin. Invest. 88, 1778-1783). These animalmodel data strongly support the expection of a therapeutic role forselectin antagonists in a wide variety of disease states in which hosttissue damage is neutrophil-mediated.

Other examples of lectins that recognize endogenous carbohydrates areCD22β, CD23, CD44 and sperm lectins (A. Varki, 1993, Glycobiol.3,97-130; P. M. Wassarman, 1988, Ann. Rev. Biochem. 57, 415-442). CD22β isinvolved in early stages of B lymphocyte activation; antagonists maymodulate the immune response. CD23 is the low affinity IgE receptor;antagonists may modulate the IgE response in allergies and asthma. CD44binds hyaluronic acid and thereby mediates cell/cell and cell/matrixadhesion; antagonists may modulate the inflammatory response. Spermlectins are thought to be involved in sperm/egg adhesion and in theacrosomal response; antagonists may be effective contraceptives, eitherby blocking adhesion or by inducing a premature, spermicidal acrosomalresponse.

Antagonists to lectins that recognise exogenous carbohydrates may havewide application for the prevention of infectious diseases. Many viruses(influenza A, B and C; Sendhi, Newcastle disease, coronavirus,rotavirus, encephalomyelitis virus, enchephalomyocarditis virus,reovirus, paramyxovirus) use lectins on the surface of the viralparticle for attachment to cells, a prerequiste for infection;antagonists to these lectins are expected to prevent infection (A.Varki, 1993, Glycobiol.3, 97-130). Similarly colonization/infectionstrategies of many bacteria utilize cell surface lectins to adhere tomammalian cell surface glyco-conjugates. Antagonists to bacterial cellsurface lectins are expected to have therapeutic potential for a widespectrum of bacterial infections, including: gastric (Helicobacterpylori), urinary tract (E.coli), pulmonary (Klebsiella pneumoniae,Stretococcus pneumoniae, Mycoplasma pneumoniae) and oral (Actinomycesnaeslundi and Actinomyces viscosus) colonization/infection (S. N.Abraham, 1994, Bacterial Adhesins, in The Handbook ofImmunopharmacology: Adhesion Molecules, C. D. Wegner, ed; B. J. Mann etal., 1991, Proc. Natl. Acad. Sci. 88, 3248-3252). A specific bacterialmediated disease state is Pseudomonas aeruginosa infection, the leadingcause of morbidity and mortality in cystic fibrosis patients. Theexpectation that high affinity antagonists will have efficacy intreating P. aeruginosa infection is based on three observations. First,a bacterial cell surface, GalNAclβ1-4Gal binding lectin mediatesinfection by adherence to asialogangliosides (αGM1 and αGM2) ofpulmonary epithelium (L. Imundo et al., 1995, Proc. Natl. Acad. Sci 92,3019-3023). Second, in vitro, the binding of P. aeruginosa is competedby the gangliosides' tetrasaccharide moiety,Galβ1-3GalNAcβ1-4Galβ1-4Glc. Third, in vivo, instillation of antibodiesto Pseudomonas surface antigens can prevent lung and pleural damage (J.F. Pittet et al., 1993, J. Clin. Invest. 92, 1221-1228).

Non-bacterial microbes that utilize lectins to initiate infectioninclude Entamoeba histalytica (a Gal specific lectin that mediatesadhesion to intestinal muscosa; W. A. Petri, Jr., 1991, AMS News57:299-306) and Plasmodium faciparum (a lectin specific for the terminalNeu5Ac(a2-3)Gal of glycophorin A of erthrocytes; P. A. Orlandi et al.,1992, J. Cell Biol. 116:901-909). Antagonists to these lectins arepotential theraputics for dysentary and malaria.

Toxins are another class of proteins that recognize exogenouscarbohydrates (K. A. Karlsson, 1989, Ann. Rev. Biochem. 58:309-350).Toxins are complex, two domain molecules, composed of a functional and acell recognition/adhesion domain. The adhesion domain is often a lectin(ie., bacterial toxins: pertussis toxin, cholera toxin, heat labiletoxin, verotoxin and tetanus toxin; plant toxins: ricin and abrin).Lectin antagonists are expected to prevent these toxins from bindingtheir target cells and consequently to be useful as antitoxins.

There are still other conditions for which the role of lectins iscurrently speculative. For example, genetic mutations result in reducedlevels of the serum mannose-binding protein (MBP). Infants who haveinsufficient levels of this lectin suffer from severe infections, butadults do not. The high frequency of mutations in both oriental andCaucasian populations suggests a condition may exist in which low levelsof serum mannose-binding protein are advantageous. Rheumatoid arthritus(RA) may be such a condition. The severity of RA is correlated with anincrease in IgG antibodies lacking terminal galactose residues on Fcregion carbohydrates (A. Young et al., 1991, Arth. Rheum. 34, 1425-1429;I. M. Roitt et al., 1988, J. Autoimm. 1, 499-506). Unlike their normalcounterpart, these gal-deficient carbohydrates are substrates for MBP.MBP/IgG immunocomplexes may contribute to host tissue damage throughcomplement activation. Similarly, the eosinophil basic protein iscytotoxic. If the cytotoxicity is mediated by the lectin activity ofthis protein, then a lectin antagonist may have therapeutic applicationsin treating eosinophil mediated lung damage.

Lectin antagonists may also be useful as imaging agents or diagnostics.For example, E-selectin antagonists may be used to image inflamedendothelium. Similarly antagonists to specific serum lectins, ie.mannose-binding protein, may also be useful in quantitating proteinlevels.

Lectins are often complex, multi-domain, multimeric proteins. However,the carbohydrate-binding activity of mammalian lectins is normally theproperty of a carbohydrate recognition domain or CRD. The CRDs ofmammalian lectins fall into three phylogenetically conserved classes:C-type, S-type and P-type (K. Drickamer and M. E. Taylor, 1993, Annu.Rev. Cell Biol. 9, 237-264). C-type lectins require Ca⁺⁺ for ligandbinding, are extracellular membrane and soluble proteins and, as aclass, bind a variety of carbohydrates. S-type lectins are most activeunder reducing conditions, occur both intraand extracellularly, bindβ-galactosides and do not require Ca⁺⁺. P-type lectins bind mannose6-phosphate as their primary ligand.

Although lectin specificity is usually expressed in terms ofmonosaccharides and/or oligosacchrides (ie., MBP binds mannose, fucoseand N-acetylglucosamine), the affinity for monosaccharides is weak. Thedissociation constants for monomeric saccharides are typically in themillimolar range (Y. C. Lee, 1992, FASEB J. 6:3193-3200; G. D. Glick etal., 1991, J Biol.Chem. 266:23660-23669; Y.Nagata and M. M.Burger, 1974,J. Biol. Chem. 249:116-3122).

Co-crystals of MBP complexed with mannose oligomers offer insight intothe molecular limitations on affinity and specificity of C-type lectins(W. I. Weis et al., 1992, Nature 360:127-134; K. Drickamer, 1993,Biochem. Soc. Trans. 21:456-459). The 3- and 4-hydroxyl groups ofmannose form coordination bonds with bound Ca⁺⁺ ion #2 and hydrogenbonds with glutamic acid (185 and 193) and asparagine (187 and 206). Thelimited contacts between the CRD and bound sugar are consistent with itsspectrum of monosaccharide binding; N-acetylglucosamine has equatorial3- and 4-hydroxyls while fucose has similarly configured hydroxyls atthe 2 and 3 positions.

The affinity of the mannose-binding protein and other lectins for theirnatural ligands is greater than that for monosaccharides. Increasedspecificity and affinity can be accomplished by establishing additionalcontacts between a protein and its ligand (K. Drickamer, 1993, supra)either by 1) additional contacts with the terminal sugar (ie., chickenhepatic lectin binds N-acetylglucose amine with greater affinity thanmannose or fucose suggesting interaction with the 2-substituent); 2)clustering of CRDs for binding complex oligosaccharides (ie., themammalian asialylglycoprotein receptor); 3) interactions with additionalsaccharide residues (ie., the lectin domain of selectins appears tointeract with two residues of the tetrasaccharide sialyl-Lewis^(X) :with the charged terminal residue, sialic acid, and with the fucoseresidue; wheat germ agglutinin appears to interact with all threeresidues of trimers of N-acetylglcosamine); or by 4) contacts with anon-carbohydrate portion of a glyco-protein.

The low affinity of lectins for mono- and oligo-saccharides presentsmajor difficulties in developing high affinity antagonists that may beuseful theraputics. Approaches that have been used to developantagonists are similar to those that occur in nature: 1) addition ormodification of substituents to increase the number of interactions; and2) multimerization of simple ligands.

The first approach has had limited success. For example, homologues ofsialic acid have been analysed for affinity to influenza virushemagglutinin (S. J. Watowich et al. 1994, Structure 2:719-731). Thedissociation constants of the best analogues are 30 to 300 μM which isonly 10 to 100-fold better than the standard monosaccharide.

Modifications of carbohydrate ligands to the selectins have also hadlimited success. In static ELISA competition assays, sialyl-Lewis^(a)and sialyl-Lewis^(X) have IC₅₀ s of 220 μM and 750 μM, respectively, forthe the inhibition of the binding of an E-selectin/lgG chimera toimmobilized sialyl-Lewis^(X) (R. M. Nelson et al., 1993, J. Clin.Invest. 91, 1157-1166). The IC₅₀ of a sialyl-Lewis^(a) derivative(addition of an aliphatic aglycone to the GlcNAc and replacement of theN-acetyl with an NH₂ group) improved 10-fold to 21 μM. Similarly,removal of the N-acetyl from sialyl-Lewis^(X) improves inhibition in anassay dependent manner (C. Foxall et al., 1992, J. Cell Biol. 117,895-902; S. A. DeFrees et al, 1993, J. Am. Chem. Soc. 115, 7549-75500.

The second approach, multimerization of simple ligands, can lead todramatic improvements in affinity for lectins that bind complexcarbohydrates (Y. C. Lee, supra). On the other hand, the approach doesnot show great enhancement for lectins that bind simpleoligosaccharides; dimerizing sialyl-Lewis^(X), a minimal carbohydrateligand for E-selectin, improves inhibition approximately 5-fold (S. A.DeFrees et al., supra).

An alternative approach is to design compounds that are chemicallyunrelated to the natural ligand. In the static ELISA competition assaysinositol polyanions inhibit L- and P-selectin binding with IC₅₀ s thatrange from 1.4 μM to 2.8 mM (O. Cecconi et al., 1994, J. Biol. Chem.269, 15060-15066). Synthetic oligopeptides, based on selectin amino acidsequences, inhibit neutrophil binding to immobilized P-selectin withIC₅₀ s ranging from 50 μM to 1 μM (J. G. Geng et al., 1992, J of Biol.Chem. 267, 19846-19853).

Lectins are nearly ideal targets for isolation of antagonists by SELEXtechnology described below. The reason is that oligonucleotide ligandsthat are bound to the carbohydrate binding site can be specificallyeluted with the relevant sugar(s). Oligonucleotide ligands withaffinities that are several orders of magnitude greater than that of thecompeting sugar can be obtained by the appropriate manipulation of theRNA to competitor ratio. Since the carbohydrate binding site is theactive site of a lectin, essentially all ligands isolated by thisprocedure will be antagonists. In addition, these SELEX ligands willexhibit much greater specifity than monomeric and oligomericsaccharides.

A method for the in vitro evolution of nucleic acid molecules withhighly specific binding to target molecules has been developed. Thismethod, Systematic Evolution of Ligands by EXponential enrichment,termed SELEX, is described in U.S. patent application Ser. No.07/536,428, entitled "Systematic Evolution of Ligands by ExponentialEnrichment", now abandoned, U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991, entitled "Nucleic Acid Ligands", now U.S. Pat. No.5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17,1992, entitled "Nucleic Acid Ligands", now U.S. Pat. No. 5,270,163 (seealso PCT/U.S. Pat. No. 91/04078), each of which is herein specificallyincorporated by reference. Each of these applications, collectivelyreferred to herein as the SELEX Patent Applications, describes afundamentally novel method for making a nucleic acid ligand to anydesired target molecule.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules, dissociating thenucleic acid-target complexes, amplifying the nucleic acids dissociatedfrom the nucleic acid-target complexes to yield a ligand-enrichedmixture of nucleic acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific, high affinity nucleic acid ligands tothe target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on theBasis of Structure", describes the use of SELEX in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 07/964,624, filed Oct. 21, 1992, entitled "Methods of ProducingNucleic Acid Ligands", now U.S. Pat. No. 5,496,938, describes methodsfor obtaining improved nucleic acid ligands after SELEX has beenperformed. U.S. patent application Ser. No. 08/123,935, filed Sep. 17,1993, entitled "Photoselection of Nucleic Acid Ligands" describes aSELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. patent application Ser.No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic AcidLigands That Discriminate Between Theophylline and Caffeine", describesa method for identifying highly specific nucleic acid ligands able todiscriminate between closely related molecules, termed Counter-SELEX.U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993,entitled "Systematic Evolution of Ligands by EXponential Enrichment:Solution SELEX", describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule. U.S. patent application Ser. No.08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution ofLigands by EXponential Enrichment: Chemi-SELEX", describes methods forcovalently linking a ligand to its target.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic AcidLigands Containing Modified Nucleotides", that describesoligonucleotides containing nucleotide derivatives chemically modifiedat the 5 and 2'-positions of pyrimidines. U.S. patent application Ser.No. 08/134,028, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2'-amino (2'-NH₂),2'-fluoro (2'-F), and/or 2-O-methyl (2-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method ofPreparation of 2' Modified Pyrimidine Intramolecular NucleophilicDisplacement", describes novel methods for making 2'-modifiednucleosides.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled "Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX" and U.S. patent application Ser. No.08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution ofLigands by Exponential Enrichment: Blended SELEX", respectively. Theseapplications allow the combination of the broad array of shapes andother properties, and the efficient amplification and replicationproperties, of oligonucleotides with the desirable properties of othermolecules. Each of the above described patent applications whichdescribe modifications of the basic SELEX procedure are specificallyincorporated by reference herein in their entirety.

The present invention applies the SELEX methodology to obtain nucleicacid ligands to lectin targets. Lectin targets, or lectins, include allthe non-enzymatic carbohydrate-binding proteins of non-immune origin,which include, but are not limited to, those described above.

Specifically, high affinity nucleic acid ligands to the GlcNAc bindinglectin, wheat germ agglutinin of Triticum vulgare have been isolated.For the purposes of the invention the terms wheat germ agglutinin, wheatgerm lectin and WGA are used interchangeably. Wheat germ agglutinin(WGA) is widely used for isolation, purification and structural studiesof glyco-conjugates.

As outlined above, the selectins are important anti-inflammatorytargets. Antagonists to the selectins modulate extravasion of leukocytesat sites of inflammation and thereby reduce neutrophil caused hosttissue damage. Using SELEX technology, isolated high affinityantagonists of L-selectin, E-selectin and P-selectin mediated adhesionare isolated.

BRIEF SUMMARY OF THE INVENTION

The present invention includes methods of identifying and producingnucleic acid ligands to lectins and the nucleic acid ligands soidentified and produced. More particularly, nucleic acid ligands areprovided that are capable of binding specifically to Wheat GermAgglutinin (WGA), L-Selectin, E-selectin and P-selectin.

Further included in this invention is a method of identifying nucleicacid ligands and nucleic acid ligand sequences to lectins comprising thesteps of (a) preparing a candidate mixture of nucleic acids, (b)partitioning between members of said candidate mixture on the basis ofaffinity to said lectin, and (c) amplifying the selected molecules toyield a mixture of nucleic acids enriched for nucleic acid sequenceswith a relatively higher affinity for binding to said lectin.

More specifically, the present invention includes the nucleic acidligands to lectins identified according to the above-described method,including those ligands to Wheat Germ Agglutinin listed in Table II, andthose ligands to L-selectin listed in Tables VIII and XII. Additionally,nucleic acid ligands to E-selectin, P-selectin and serum mannose bindingprotein are provided. Also included are nucleic acid ligands to lectinsthat are substantially homologous to any of the given ligands and thathave substantially the same ability to bind lectins and antagonize theability of the lectin to bind carbohydrates. Further included in thisinvention are nucleic acid ligands to lectins that have substantiallythe same structural form as the ligands presented herein and that havesubstantially the same ability to bind lectins and antagonize theability of the lectin to bind carbohydrates.

The present invention also includes modified nucleotide sequences basedon the nucleic acid ligands identified herein and mixtures of the same.

The present invention also includes the use of the nucleic acid ligandsin therapeutic, prophylactic and diagnostic applications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows consensus hairpin secondary structures for WGA 2'NH₂ RNAligands: (a) family 1, (b) family 2, and (c) family 3. Nucleotidesequence is in standard one letter code. Invariant nucleotides are inbold type. Nucleotides derived from fixed sequence are in lower case.

FIG. 2 shows binding curves for the L-selectin SELEX second and ninthround 2'NH₂ RNA pools to peripheral blood lymphocytes (PBMCs).

FIG. 3 shows binding curves for random 40N7 2'NH₂ RNA (SEQ. ID NO: 64)and the cloned L-selectin ligand, F14.12 (SEQ. ID NO: 78), to peripheralblood lymphocytes (PBMC).

FIG. 4 shows the results of a competition experiment in which thebinding of 5 nM ³² P-labeled F14.12 (SEQ. ID NO: 78) to PBMCs (10⁷ /ml)is competed with increasing concentrations of unlabeled F14.12 (SEQ. IDNO: 78). RNA Bound equals 100 ×(net counts bound in the presence ofcompetitor/net counts bound in the absence of competitor).

FIG. 5 shows the results of a competition experiment in which thebinding of 5 nM ³² P-labeled F14.12 (SEQ. ID NO: 78) to PBMCs (10⁷ /ml)is competed with increasing concentrations of the blocking monoclonalanti-L-selectin antibody, DREG-56, or an isotype matched, negativecontrol antibody. RNA Bound equals 100×(net counts bound in the presenceof competitor/net counts bound in the absence of competitor).

FIG. 6 shows the results of a competitive ELISA assay in which thebinding of soluble LS-Rg to immobilized sialyl-Lewis^(X) /BSA conjugatesis competed with increasing concentrations of unlabeled F14.12 (SEQ. IDNO: 78). Binding of LS-Rg was monitored with an HRP conjugatedanti-human IgG antibody. LS-Rg Bound equals 100×(OD₄₅₀ in the presenceof competitor)/(OD₄₅₀ in the absence of competitor). The observed OD₄₅₀was corrected for nonspecific binding by subtracting the OD₄₅₀ in theabsence of LS-Rg from the experimental values. In the absence ofcompetitor the OD₄₅₀ was 0.324 and in the absence of LS-Rg 0.052.Binding of LS-Rg requires divalent cations; in the absence ofcompetitor, replacement of Ca⁺⁺ /Mg⁺⁺ with 4 mM EDTA reduced the OD₄₅₀to 0.045.

FIG. 7 shows hairpin secondary structures for representative L-selectin2'NH₂ RNA ligands: (a) F13.32 (SEQ. ID NO: 67), family I (b) 6.16 (SEQ.ID NO: 84), family III and (c) F14.12 (SEQ. ID NO: 78), family II.Nucleotide sequence is in standard one letter code. Invariantnucleotides are in bold type. Nucleotides derived from fixed sequenceare in lower case.

FIG. 8 shows binding curves for the L-selectin SELEX fifteenth roundssDNA pool to PBMCs (10⁷ /ml).

FIG. 9 shows the results of a competition experiment in which thebinding of 2 nM ³² P-labeled round 15 ssDNA to PBMCs (10⁷ /ml) iscompeted with increasing concentrations of the blocking monoclonalanti-L-selectin antibody, DREG-56, or an isotype matched, negativecontrol antibody. RNA Bound equals 100×(net counts bound in the presenceof competitor/net counts bound in the absence of competitor).

FIG. 10 shows the consensus hairpin secondary structures for family 1ssDNA ligands to L-selectin. Nucleotide sequence is in standard oneletter code. Invariant nucleotides are in bold type. The base pairs athighly variable positions are designated N-N'. To the right of the stemis a matrix showing the number of occurances of particular base pairsfor the position in the stem that is on the same line.

DETAILED DESCRIPTION OF THE INVENTION

This application describes high-affinity nucleic acid ligands to lectinsidentified through the method known as SELEX. SELEX is described in U.S.patent application Ser. No. 07/536,428, entitled Systematic Evolution ofLigands by EXponential Enrichment, now abandoned, U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled NucleicAcid Ligands, U.S. patent application Ser. No. 07/931,473, filed Aug.17, 1992, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,270,163,(see also PCT/U.S. Pat. No. 91/04078). These applications, eachspecifically incorporated herein by reference, are collectively calledthe SELEX Patent Applications.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately. 05-50%) areretained during partitioning.

4) Those nucleic acids selected during partitioning as having therelatively higher affinity to the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield acandidate mixture containing one or a small number of unique nucleicacids representing those nucleic acids from the original candidatemixture having the highest affinity to the target molecule.

The SELEX Patent Applications describe and elaborate on this process ingreat detail. Included are targets that can be used in the process;methods for partitioning nucleic acids within a candidate mixture; andmethods for amplifying partitioned nucleic acids to generate enrichedcandidate mixture. The SELEX Patent Applications also describe ligandsobtained to a number of target species, including both protein targetswhere the protein is and is not a nucleic acid binding protein.

SELEX provides high affinity ligands of a target molecule. Thisrepresents a singular achievement that is unprecedented in the field ofnucleic acids research. The present invention applies the SELEXprocedure to the specific target of lectins. Specifically, the presentinvention describes the identification of nucleic acid ligands to WheatGerm Agglutinin, and the Selectins, specifically, L-selectin, E-selectinand P-selectin. In the Example section below, the experimentalparameters used to isolate and identify the nucleic acid ligands toWheat Germ Agglutinin, L-selectin, E-selectin and P-selectin aredescribed.

In order to produce nucleic acids desirable for use as a pharmaceutical,it is preferred that the nucleic acid ligand (1) binds to the target ina manner capable of achieving the desired effect on the target; (2) beas small as possible to obtain the desired effect; (3) be as stable aspossible; and (4) be a specific ligand to the chosen target. In mostsituations, it is preferred that the nucleic acid ligand have thehighest possible affinity to the target.

In the present invention, a SELEX experiment was performed in search ofnucleic acid ligands with specific high affinity for Wheat GermAgglutinin from a degenerate library containing 50 random positions(50N). This invention includes the specific nucleic acid ligands toWheat Germ Agglutinin shown in Table II (SEQ ID NOS: 4-55), identifiedby the methods described in Examples 1 and 2. The scope of the ligandscovered by this invention extends to all nucleic acid ligands of WheatGerm Agglutinin, modified and unmodified, identified according to theSELEX procedure. More specifically, this invention includes nucleic acidsequences that are substantially homologous to the ligands shown inTable II. By substantially homologous it is meant a degree of primarysequence homology in excess of 70%, most preferably in excess of 80%. Areview of the sequence homologies of the ligands of Wheat GermAgglutinin shown in Table II shows that sequences with little or noprimary homology may have substantially the same ability to bind WheatGerm Agglutinin. For these reasons, this invention also includes nucleicacid ligands that have substantially the same ability to bind Wheat GermAgglutinin as the nucleic acid ligands shown in Table II. Substantiallythe same ability to bind Wheat Germ Agglutinin means that the affinityis within a few orders of magnitude of the affinity of the ligandsdescribed herein. It is well within the skill of those of ordinary skillin the art to determine whether a given sequence--substantiallyhomologous to those specifically described herein--has substantially thesame ability to bind Wheat Germ Agglutinin.

In the present invention, a SELEX experiment was performed in search ofnucleic acid ligands with specific high affinity for L-selectin from adegenerate library containing 40 random positions (40N). This inventionincludes the specific nucleic acid ligands to L-selectin shown in TablesVIII and XII (SEQ ID NOS: 67-117 and 129-167), identified by the methodsdescribed in Examples 7, 8, 13 and 14. The scope of the ligands coveredby this invention extends to all nucleic acid ligands of L-selectin,modified and unmodified, identified according to the SELEX procedure.More specifically, this invention includes nucleic acid sequences thatare substantially homologous to the ligands shown in Tables VIII andXII. By substantially homologous it is meant a degree of primarysequence homology in excess of 70%, most preferably in excess of 80%. Areview of the sequence homologies of the ligands of L-selectin shown inTables VIII and XII shows that sequences with little or no primaryhomology may have substantially the same ability to bind L-selectin. Forthese reasons, this invention also includes nucleic acid ligands thathave substantially the same ability to bind L-selectin as the nucleicacid ligands shown in Tables VII and XII. Substantially the same abilityto bind L-selectin means that the affinity is within a few orders ofmagnitude of the affinity of the ligands described herein. It is wellwithin the skill of those of ordinary skill in the art to determinewhether a given sequence--substantially homologous to those specificallydescribed herein--has substantially the same ability to bind L-selectin.

In the present invention, a SELEX experiment was performed in search ofnucleic acid ligands with specific high affinity for E-selectin andP-selectin from a degenerate library containing 40 random positions(40N). This invention includes specific nucleic acid ligands toE-selectin and P-selectin identified by the methods described inExamples 19 and 20. The scope of the ligands covered by this inventionextends to all nucleic acid ligands of E-selectin and P-selectin,modified and unmodified, identified according to the SELEX procedure.

This invention also includes the ligands as described above, whereincertain chemical modifications are made in order to increase the in vivostability of the ligand or to enhance or mediate the delivery of theligand. Examples of such modifications include chemical substitutions atthe sugar and/or phosphate and/or base positions of a given nucleic acidsequence. See, e.g., U.S. patent application Ser. No. 08/117,991, filedSep. 9, 1993, entitled High Affinity Nucleic Acid Ligands ContainingModified Nucleotides which is specifically incorporated herein byreference. Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process. Additionally, the nucleic acid ligands of the inventioncan be complexed with various other compounds, including but not limitedto, lipophilic compounds or nonimmunogenic, high molecular weightcompounds. Lipophilic compounds include, but are not limited to,cholesterol, dialkyl glycerol, and diacyl glycerol. Non-immunogenic,high molecular weight compounds include, but are not limited to,polyethylene glycol, dextran, albumin and magnetite. This type ofnucleic acid ligand complex is described in copending U.S. patentapplication Ser. Number 08/434,465, filed May 4, 1995, entitled "NucleicAcid Ligand Complexes", which is herein incorporated by reference.

Well established animal models exist for many of the disease stateswhich are candidates for selectin antagonist therapy. Models availablefor testing the efficacy of oligonucleotide selectin antagonistsinclude:

1) mouse models for peritoneal inflammation (P. Pizcueta and F. W.Luscinskas, 1994, Am. J. Pathol. 145, 461-469), diabetes (A. C. Hanninenet al., 1992, J. Clin. Invest. 92, 2509-2515), lymphocyte trafficking(L. M. Bradley et al, 1994, J. Exp. Med, 2401-2406), glomerulonephritis(P. G. Tipping et al., 1994k Kidney Int. 46, 79-88), experimentalallergic encephalomyelitis (J. M. Dopp et al., 1994, J. Neuroimmunol.54: 129-144), acute inflammation in human/SCID mouse chimera (H. C. Yanet al., 1994, J. Immunol. 152, 3053-3063), endotoxin-mediatedinflammation (W. E. Sanders et al., 1992, Blood 80, 795 800);

2) rat models for acute lung injury (M. S. Milligan et al., 1994, J.Immunol. 152, 832-840), hind limb ischemia/reperfusion injury (A.Seekamp et al., 1994, Am. J. Pathol 144, 592-598), remote lung injury(A. Seekamp et al., 1994, supra; D. L. Carden et al., 1993, J. Appl.Physiol 75, 2529-2543), neutrophil rolling on mesenteric venules (K. Leyet al., 1993, Blood 82, 132-1638), myocardial infarction ischemiareperfusion injury (D. Altavilla et al., 1994, Eur. J. Pharmacol.Environ. Toxicol. Pharmacol. 270, 45-51);

3) rabbit models for hemorrhagic shock (R. K. Winn et al., 1994, Am. J.Physiol. Heart Circ. Physiol. 267, H2391-H2397), ear ischemiareperfusion injury (D. Mihelcic et al., 1994, Bollod 84, 2333-2328neutrophil rolling on mesenteric venules (A. M. Olofsson et al., Blood84, 2749-2758), experimental meningitis (C. Granert et al., 1994, J.Clin. Invest. 93, 929-936); lung, peritoneal and subcutaneous bacterialinfection (S. R. Sharer et al., 1993, J. Immunol. 151, 4982 4988),myocardial ischemia/repefusion (G. Montrucchio et al., 1989, Am. J.Physiol. 256, H1236-H1246), central nervous system ischemic injury (W.M. Clark et al., 1991, Stroke 22, 877-883);

4) cat models for myocardial infraction ischemia reperfusion injury(M.Buerke et al., 1994, J. Pharmacol. Exp. Ther. 271, 134-142);

5) dog models for myocardial infarction ischemia reperfusion injury(D.J. Lefer et al., 1994, Circulation 90, 2390-2401);

6) pig models for arthritis (F. Jamar et al., 1995, Radiology 194,843-850);

7) rhesus monkey models for cutaneous inflammation (A. Silber et al.,Lab. Invest. 70, 163-175);

8) cynomolgus monkey models for renal transplants (S. L. Wee, 1991,Transplant. Prod. 23, 279-280); and

9) baboon models for dacron grafts (T. Palabrica et al, 1992, Nature359, 848-851), septic, traumatic and hypovoemic shock (H. Redl et al.,1991, Am. J. Pathol. 139, 461-466).

The nucleic acid ligands to lectins described herein are useful aspharmaceuticals and as diagnostic reagents.

EXAMPLES

The following examples are illustrative of certain embodiments of theinvention and are not to be construed as limiting the present inventionin any way. Examples 1-6 describe identification and characterization of2'NH₂ RNA ligands to Wheat Germ Agglutinin. Examples 7-12 describedidentification and characterization of 2'NH₂ RNA ligands to L-selectin.Examples 13-17 describe identification and characterization of ssDNAligands to L-selectin. Example 18 describes identification of nucleicacid ligands to E-selectin. Example 19 describes identification ofnucleic acid ligands to P-selectin.

EXAMPLE 1 Nucleic Acid Ligands to Wheat Germ Agglutinin

Experimental Procedures

A) Materials

Wheat Germ Lectin (Triticum vulgare) Sepharose 6MB beads were purchasedfrom Pharmacia Biotech. Free Wheat Germ Lectin (Triticum vulgare) andall other lectins were obtained from E Y Laboratories;methyl-α-D-mannopyranoside was from Calbiochem andN-acetyl-D-glucosamine, GlcNAc, and the trisaccharide NN'N'-triacetylchitotriose, (GlcNAc)₃, were purchased from Sigma ChemicalCo. The 2'NH₂ modified CTP and UTP were prepared according to Pieken et.al. (1991, Science 253:314-317). DNA oligonucleotides were synthesizedby Operon. All other reagents and chemicals were purchased fromcommercial sources. Unless otherwise indicated, experiments utilizedHanks' Balanced Salt Solutions (HBSS; 1.3 mM CaCl₂, 5.0 mM KCI, 0.3 mMKH₂ PO₄, 0.5 mM MgCl₂.6H₂ O, 0.4 mM MgSO₄.7H₂ O, 138 mM NaCl, 4.0 mMNaHCO₃, 0.3 mM Na₂ HPO4, 5.6 mM D-Glucose; GibcoBRL).

B) SELEX

The SELEX procedure is described in detail in U.S. Pat. No. 5,270,163and elsewhere. In the wheat germ agglutinin SELEX experiment, the DNAtemplate for the initial RNA pool contained 50 random nucleotides,flanked by N9 5' and 3' fixed regions (50N9) 5'gggaaaagcgaaucauacacaaga-50N-gcuccgccagagaccaaccgagaa 3' (SEQ ID NO: 1).All C and U have 2'NH₂ substituted for 2'OH for ribose. The primers forthe PCR were the following: 5' Primer 5'taatacgactcactatagggaaaagcgaatcatacacaaga 3' (SEQ ID NO: 2) and 3'Primer 5' ttctcggttggtctctggcggagc 3' (SEQ ID NO: 3). The fixed regionsof the starting random pool include DNA primer annealing sites for PCRand cDNA synthesis as well as the consensus T7 promoter region to allowin vitro transcription. These single-stranded DNA molecules wereconverted into double-stranded transcribable templates by PCRamplification. PCR conditions were 50 mM KCI, 10 mM Tris-Cl, pH 8.3,0.1% Triton X100, 7.5 mM MgCl₂, 1 mM of each dATP, dCTP, dGTP, and dTTP,and 25 U/ml of Taq DNA polymerase. Transcription reactions contained 5mM DNA template, 5 units/μl T7 RNA polymerase, 40 mM Tris-Cl (pH 8.0),12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 0.002Triton X-100, 4% PEG 8000,2 mM each of 2'OH ATP, 2'OH GTP, 2'NH₂ CTP, 2'NH₂ UTP, and 0.31 mM α-³²P 2'OH ATP.

The strategy for partitioning WGA/RNA complexes from unbound RNA was 1)to incubate the RNA pool with WGA immobilized on sepharose beads; 2) toremove unbound RNA by extensive washing; and 3) to specifically eluteRNA molecules bound at the carbohydrate binding site by incubating thewashed beads in buffer containing high concentrations of (GlcNAc)₃. TheSELEX protocol is outlined in Table 1.

The WGA density on Wheat Germ Lectin Sepharose 6MB beads isapproximately 5 mg/ml of gel or 116 μM (manufacturer's specifications).After extensive washing in HBSS, the immobilized WGA was incubated withRNA at room temperature for 1 to 2 hours in a 2 ml siliconized columnwith constant rolling (Table I). Unbound RNA was removed by extensivewashing with HBSS. Bound RNA was eluted as two fractions; first,nonspecifically eluted RNA was removed by incubating and washing with 10mM methyl-α-D-mannopyranoside in HBSS (Table I; rounds 1-4) or with HBSS(Table I; rounds 5-11); second, specifically eluted RNA was removed byincubating and washing with 0.5 to 10 mM (GlcNAc)₃ in HBSS (Table I).The percentage of input RNA that was specifically eluted is recorded inTable I.

The specifically eluted fraction was processed for use in the followinground. Fractions eluted from immobilized WGA were heated at 90° C. for 5minutes in 1% SDS, 2% β-mercaptoethanol and extracted withphenol/chloroform. RNA was reverse transcribed into cDNA by AMV reversetranscriptase at 48° C. for 60 min in 50 mM Tris-Cl pH (8.3), 60 mMNaCl, 6 mM Mg(OAc)₂, 10 mM DTT, 100 pmol DNA primer, 0.4 mM each ofdNTPs, and 0.4 unit/μl AMV RT. PCR amplification of this cDNA resultedin approximately 500 pmol double-stranded DNA, transcripts of which wereused to initiate the next round of SELEX.

D) Nitrocellulose Filter Partitioning

As described in SELEX Patent Applications, the nitrocellulose filterpartitioning method was used to determine the affinity of RNA ligandsfor WGA and for other proteins. Filter discs (nitrocellulose/celluloseacetate mixed matrix, 0.45 μm pore size, Millipore; or purenitrocellulose, 0.45 μm pore size, Bio-Rad) were placed on a vacuummanifold and washed with 4 ml of HBSS buffer under vacuum. Reactionmixtures, containing ³² p labeled RNA pools and unlabeled WGA, wereincubated in HBSS for 10 min at room temperature, filtered, and thenimmediately washed with 4 ml HBSS. The filters were airdried and countedin a Beckman LS6500 liquid scintillation counter without fluor. WGA is ahomodimer, molecular weight 43.2 kD, with 4 GlcNAc binding sites perdimer. For affinity calculations, we assume one RNA ligand binding siteper monomer (two per dimer). The monomer concentration is defined as 2times the dimer concentration. The equilibrium dissociation constant,K_(d), for an RNA pool or specific ligand that binds monophasically isgiven by the equation

    K.sub.d = P.sub.f ! R.sub.f ! R.sub.P !

where, Rf!=free RNA concentration

Pf!=free WGA monomer concentration

RP!=concentration of RNA/WGA monomer complexes

K_(D) =dissociation constant

A rearrangement of this equation, in which the fraction of RNA bound atequilibrium is expressed as a function of the total concentration of thereactants, was used to calculate Kds of monophasic binding curves:

    q=(P.sub.T +R.sub.T +K.sub.D -((P.sub.T +R.sub.T +K.sub.D).sup.2 -4P.sub.T R.sub.T).sup.1/2)

q=fraction of RNA bound

P_(T!) =total WGA monomer concentration

R_(T!) =total RNA concentration K_(D) S were determined by least squarefitting of the data points using the graphics program Kaleidagraph(Synergy Software, Reading, PA).

E) Cloning and Sequencing contain a BamH1 or a EcoR1 restrictionendonuclease recognition site. Using these restriction sites the DNAsequences were inserted directionally into the pUC 18 vector. Theserecombinant plasmids were transformed into E. coli strain JM109(Stratagene, La Jolla, Calif.). Plasmid DNA was prepared according tothe alkaline hydrolysis method (Zhou et al., 1990 Biotechniques8:172-173) and about 72 clones were sequenced using the Sequenaseprotocol (United States Biochemical Corporation, Cleveland, Ohio). Thesequences are provided in Table II.

F) Competitive Binding Studies

Competitive binding experiments were performed to determine if RNAligands and (GIcNAc)₃ bind the same site on WGA. A set of reactionmixtures containing α ³² P labeled RNA ligand and unlabeled WGA, each ata fixed concentration (Table V), was incubated in HBSS for 15 min atroom temperature with (GlcNAc)₃. Individual reaction mixtures were thenincubated with a (GlcNAc)₃ dilution from a 2-fold dilution series for 15minutes. The final (GlcNAc)₃ concentrations ranged from 7.8 μM to 8.0 mM(Table V). The reaction mixtures were filtered, processed and counted asdescribed in "Nitrocellulose Filter Partitioning", paragraph D above.

Competition titration experiments were analyzed by the followingequation to determine the concentration of free protein P! as a functionof the total concentration of competitor added, C_(T) !:

    0= P!(1+K.sub.L  L.sub.T !/(1+K.sub.L  P!)+K.sub.C  C.sub.T !/(1+K.sub.C  P!))-P.sub.T

where L_(T) is the concentration of initial ligand, K_(L) is the bindingconstant of species L to the protein (assuming 1:1 stiochiometry) and Kcis the binding constant of species C to the protein (assuming 1:1stiochiometry). Since it is difficult to obtain a direct solution forequation 1, iteration to determine values of P! to a precision of1×10⁻¹⁵ was used. Using these values of P!, the concentration ofprotein-ligand complex P_(L) ! as a function of C_(T) ! was determinedby the following equation:

     PL!=K.sub.L  L.sub.T ! P!(1+K.sub.L  P!)

Since the experimental data is expressed in terms of % PL!, thecalculated concentration of PL! was normalized by the initialconcentration of PL_(o) ! before addition of the competitor. ( PL_(o) !was calculated using the quadratic solution for the standard bindingequation for the conditions used (ref)). The maximum (M) and minimum (B)% PL! was allowed to float during the analysis as shown in the followingequation.

    % PL!= PL!/ PL.sub.o !*(M-B)+B

A non-linear least-squares fitting procedure was used as described by P.R. Bevington (1969) Data Reduction and Error Analysis for the PhysicalSciences, McGraw-Hill publishers. The program used was originallywritten by Stanley J. Gill in MatLab and modified for competitionanalysis by Stanley C. Gill. The data were fit to equations 1-3 toobtain best fit parameters for K_(c), M and B as a function of C_(T) !while leaving K_(L) and P_(T) fixed.

G) Inhibition of WGA Agglutinating Activity

Agglutination is a readily observed consequence of the interaction of alectin with cells and requires that individual lectin moleculescrosslink two or more cells. Lectin mediated agglutination can beinhibited by sugars with appropriate specificity. Visual assay of thehemagglutinating activity of WGA and the inhibitory activity of RNAligands, GIcNAc and (GlcNAc)₃ was made in Falcon round bottom 96 wellmicrotiter plates, using sheep erythrocytes. Each well contained 54 μlof erythrocytes (2.5×10⁸ cells/ml) and 54 μl of test solution.

To titrate WGA agglutinating activity, each test solution contained aWGA dilution from a 4-fold dilution series. The final WGA concentrationsranged from 0.1 pM to 0.5 μM. For inhibition assays, the test solutionscontained 80 nM WGA (monomer) and a dilution from a 4-fold dilutionseries of the designated inhibitor. Reaction mixtures were incubated atroom temperature for 2 hours, after which time no changes were observedin the precipitation patterns of erythrocytes. These experiments werecarried out in Gelatin Veronal Buffer (0.15 mM CaCl₂, 141 mM NaCl, 0.5mM Mg Cl₂, 0.1% gelatin, 1.8 mM sodium barbial, and 3.1 mM barbituricacid, pH 7.3-7.4; Sigma #G-6514).

In the absence of agglutination, erythrocytes settle as a compactpellet. Agglutinated cells form a more diffuse pellet. Consequently, invisual tests, the diameter of the pellet is diagnostic foragglutination. The inhibition experiments included positive and negativecontrols for agglutination and appropriate controls to show that theinhibitors alone did not alter the normal precipitation pattern.

EXAMPLE 2 RNA Ligands to WGA

A. SELEX

The starting RNA library for SELEX, randomized 50N9 (SEQ ID NO: 1),contained approximately 2×10¹⁵ molecules (2 nmol RNA). The SELEXprotocol is outlined in Table I. Binding of randomized RNA to WGA isundetectable at 36 μM WGA monomer. The dissociation constant of thisinteraction is estimated to be >4 mM .

The percentage of input RNA eluted by (GlcNAc)₃ increased from 0.05% inthe first round, to 28.5% in round 5 (Table I). The bulk K_(d) of round5 RNA was 600 nM (Table I). Since an additional increase in specificallyeluted RNA was not observed in round 6a (Table I), round 6 was repeated(Table I, round 6b) with two modifications to increase the stringency ofselection: the volume of gel, and hence the mass of WGA, was reduced tenfold; and RNA was specifically eluted with increasing concentrations of(GlcNAc)₃, in stepwise fashion, with only the last eluted RNA processedfor the following round. The percentage of specifically eluted RNAincreased from 5.7% in round 6b to 21.4% in round 8, with continuedimprovement in the bulk K_(d) (260 nM, round 8 RNA, Table I).

For rounds 9 through 11, the WGA mass was again reduced ten fold tofurther increase stringency. The K_(d) of round 11 RNA was 68 nM .Sequencing of the bulk starting RNA pool and sixth and eleventh roundRNA revealed some nonrandomness in the variable region at the sixthround and increased nonrandomess at round eleven. To monitor the progessof SELEX, ligands were cloned and sequenced from round 6b and round 11.From each of the two rounds, 36 randomly picked clones were sequenced.Sequences were aligned manually and are shown in Table II.

B. RNA Sequences

From the sixth and eleventh rounds, respectively, 27 of 29 and 21 of 35sequenced ligands were unique. The number before the "." in the ligandname indicates whether it was cloned from the round 6 or round 11 pool.Only a portion of the entire clone is shown in Table II (SEQ ID NOS:4-55). The entire evolved random region is shown in upper case letters.Any portion of the fixed region is shown in lower case letters. Bydefinition, each clone includes both the evolved sequence and theassociated fixed region, unless specifically stated otherwise. A uniquesequence is operationally defined as one that differs from all others bythree or more nucleotides. In Table II, ligands sequences are shown instandard single letter code (Comish-Bowden, 1985 NAR 13: 3021-3030).Sequences that were isolated more than once are indicated by theparenthetical number, (n), following the ligand isolate number. Theseclones fall into nine sequence families (1-9) and a group of unrelatedsequences (Orphans).

The distribution of families from round six to eleven provides a clearillustration of the appearance and disappearnce of ligand families inresponse to increased selective pressure (Table II). Family 3predominant (11/29 ligands) in round 6, has nearly disappeared (2/35) byround 11. Similarly, minor familes 6 through 9 virtually disappear. Inconstrast, only one (family 1) of round eleven's predominant families(1, 2, 4 and 5) was detected in round six. The appearance anddisappearance of families roughly correlates with their bindingaffinities.

Alignment (Table II) defines consensus sequences for families 1-4 and6-9 (SEQ ID NOS: 56-63). The consensus sequences of families 1-3 arelong (20, 16 and 16, respectively) and very highly conserved. Theconsensus sequences of families 1 and 2 contain two sequences in common:the trinucleotide TCG and the pentanucleotide ACGAA. A relatedtetranucleotide, AACG, occurs in family 3. The variation in position ofthe consensus sequences within the variable regions indicates that theligands do not require a specific sequence from either the 5' or 3'fixed region.

The consensus sequences of family 1 and 2 are flanked by complementarysequences 5 or more nucleotides in length. These complementary sequencesare not conserved and the majority include minor discontinuities. Family3 also exhibits flanking complementary sequences, but these are morevariable in length and structure and utilize two nucleotide pairs ofconserved sequence.

Confidence in the family 4 consensus sequence (Table II) is limited bythe small number of ligands, the variability of spacing and the high Gcontent. The pentanucleotide, RCTGG, also occurs in families 5 and 8.Ligands of family 5 show other sequence similarities to those of family4, especially to ligand 11.28.

C. Affinities

The dissociation constants for representative members of families 1-9and orphan ligands were determined by nitrocellulose filter bindingexperiments and are listed in Table III. These calculations assume oneRNA ligand binding site per WGA monomer. At the highest WGAconcentration tested (36 μM WGA monomer), binding of random RNA is notobserved, indicating a K_(d) at least 100-fold higher than the proteinconcentration or >4 mM.

The data in Table III define several characteristics of ligand binding.First, RNA ligands to WGA bind monophasically. Second, the range ofmeasured dissociation constants is 1.4 nM to 840 nM . Third, the bindingfor a number of ligands, most of which were sixth round isolates, wasless than 5% at the highest WGA concentration tested. The dissociationconstants of these ligands are estimated to be greater than 20 μM.Fourth, on average eleventh round isolates have higher affinity thanthose from the sixth round. Fifth, the SELEX probably was not taken tocompletion; the best ligand (11.20)(SEQ ID NO: 40) is not the dominantspecies. Since the SELEX was arbitrarily stopped at the 11th round, itis not clear that 11.20 would be the ultimate winner. Sixth, even thoughthe SELEX was not taken to completion, as expected, RNA ligands wereisolated that bind WGA with much greater affinity than do mono- oroligosaccharides (ie., the affinty of 11.20 is 5×10⁵ greater than thatof GlcNAc, Kd=760 μM, and 850 better than that of (GIcNAc)₃, Kd=12 M; Y.Nagata and M. Burger, 1974, supra). This observation validates ourcontention that competitive elution allows the isolation ofoligonucleotide ligands with affinities that are serveral orders ofmagnitude greater than that of the competing sugar.

In addition these data show that even under conditions of high targetdensity, 116 pmole WGA dimer/μl of beads, it is possible to overcomeavidity problems and recover ligands with nanomolar affinities. From thesixth to the eleventh round (Table II), in response to increasedselective pressure as indicated by the improvent in bulk K_(d) (TableI), sequence families with lower than average affinity (Table III) areeliminated from the pool.

EXAMPLE 3 Specificity of RNA Ligands

The affinity of WGA ligands 6.8, 11.20 and 11.24 (SEQ ID NOS: 13, 40,and 19) for GlcNAc binding lectins from Ulex europaeus, Daturastramonium and Canavalia ensiformis were determined by nitrocellulosepartitioning. The results of this determination are shown in Table IV.The ligands are highly specific for WGA. For example, the affinity ofligand 11.20 for WGA is 1,500, 8,000 and >15,000 fold greater than it isfor the U. europaeus, D. stramonium and C. ensiformis lectins,respectively. The 8,000 fold difference in affinity for ligand 11.20exhibited by T. vulgare and D. stramonium compares to a 3 to 10 folddifference in their affinity for oligomers of GlcNAc and validates ourcontention that competitive elution allows selection of oligonucleotideligands with much greater specificty than monomeric and oligomericsaccharides (J. F. Crowley et al., 1984, Arch. Biochem. and Biophys.231:524-533; Y. Nagata and M. Burger, 1974, supra; J. P. Privat et al.,FEBS Letters 46:229-232).

EXAMPLE 4 Competitive Binding Studies

If an RNA ligand and a carbohydrate bind a common site, then binding ofthe RNA ligand is expected to be competively inhibited by thecarbohydrate. Furthermore, if the oligonucleotide ligands bindexclusively to carbohydrate binding sites, inhibition is expected to becomplete at high carbohydrate concentrations. In the experimentsreported in Table V, dilutions of unlabeled (GlcNAc)₃, from a 2-folddilution series, were added to three sets of binding reactions thatcontained WGA and an α-³² P labeled RNA ligand (6.8, 11.20 or 11.24 (SEQID NOS: 13, 40 and19); RNA! final= WGA!final=15 nM ). After a 15 minuteincubation at room temperature, the reactions were filtered andprocessed as in standard binding experiments.

Qualitatively, it is clear that RNA ligands bind only to sites at which(GlcNAc)₃ binds, since inhibition is complete at high (GlcNAc)₃concentrations (Table V). These data do not rule out the possiblity that(GlcNAc)₃ binds one or more sites that are not bound by these RNAligands.

Quantitatively, these data fit a simple model of competitive inhibition(Table V) and give estimates of 8.4, 10.9 and 19.4 μM for the Kd of(GIcNAc)₃. These estimates are in good agreement with literature values(12 μM @ 4 C, Nagata and Burger, 1974, supra; 11 μM @ 10.8 C, VanLandschoot et al., 1977, Eur. J. Biochem. 79:275-283; 50 μM, M. Monsignyet al., 1979, Eur J. Biochem. 98:39-45). These data confirm theassertion that competitive elution with a specific carbohydrate targetsthe lectin's carbohydrate binding site.

EXAMPLE 5 Inhibition of WGA Agglutinating Activity

At 0.5 μM, RNA ligands 6.8 and 11.20 (SEQ ID NO: 13 and 40) completelyinhibit WGA mediated agglutination of sheep erythrocytes (Table VI).Ligand 11.24 (SEQ ID NO: 19) is not as effective, showing only partialinhibition at 2 μM, the highest concentration tested (Table VI).(GlcNAc)₃ and GIcNAc completely inhibit agglutination at higherconcentrations, 8 μM and 800 μM, respectively, (Table VI; Monsigny etal., supra). The inhibition of agglutination varifies the contentionthat ligands isolated by this procedure will be antagonists of lectinfunction. Inhibition also suggests that more than one RNA ligand isbound per WGA dimer, since agglutination is a function of multiplecarbohydrate binding sites.

An alternative interpretation for the inhibition of agglutination isthat charge repulsion prevents negatively charged WGA/RNA complexes frombinding carbohydrates (a necessary condition for agglutination) onnegatively charged cell surfaces. This explanation seems unlikely fortwo reasons. First, negatively charged oligonucleotide ligands selectedagainst an immobilized purified protein are known to bind to the proteinwhen it is presented in the context of a cell surface (see Example #10,L-selectin cell binding). Second, negatively charged (pI=4) succinylatedWGA is as effective as native WGA (pI=8.5) in agglutinating erthrocytes(M. Monsigny et al., supra).

EXAMPLE 6 Secondary Structure of High Affinity WGA Ligands

In favorable instances, comparative analysis of aligned sequences allowsdeduction of secondary structure and structure function relationships.If the nucleotides at two positions in a sequence covary according toWatson-Crick base pairing rules, then the nucleotides at these positionsare apt to be paired. Nonconserved sequences, especially those that varyin length are not apt to be directly involved in function, while highlyconserved sequence are likely to be directly involved.

Comparative analyses of both family 1 and 2 sequences each yield ahairpin structure with a large, highly conserved loop (FIG. 1a and 1b).Interactions between loop nucleotides are likely but they are notdefined by these data. The stems of individual ligands vary in sequence,length and structure (ie., a variety of bulges and internal loops areallowed; Table II). Qualitatively it is clear that the stems arevalidated by Watson/Crick covariation and that by the rules ofcomparative analysis the stems are not directly involved in binding WGA.Family 3 can form a similar hairpin in which 2 pairs of conservednucleotides are utilized in the stem (FIG. 1c).

If it is not possible to fold the ligands of a sequence family intohomologous structures, their assignment to a single family isquestionable. Both ligand 11.7, the dominant member of family 4, andligand 11.28 can be folded into two plane G-quartets. However, thisassignment is speculative: 1) 11.28 contains five GG dinucleotides andone GGGG tetranucleotide allowing other G-quartets; and 2) ligands 11.2and 11.33 cannot form G-quartets. On the other hand, all ligands canform a hairpin with the conserved sequence GAGRFTNCRT in the loop.However, the conserved sequence RCTGGC (Table II) does not have aconsistent role in these hairpins.

Multiple G-quartet structures are possible for Family 5. One of theseresembles the ligand 11.7 G-quartet. No convincing hairpin structuresare possible for ligand 11.20.

EXAMPLE 7 2'NH₂ RNA Ligands to Human L-Selectin

Experimental Procedures

A) Materials

LS-Rg is a chimeric protein in which the extracellular domain of humanL-selectin is joined to the Fc domain of a human G2 immunoglobulin(Norgard et al., 1993, PNAS 90:1068-1072). ES-Rg, PS-Rg and CD22β-Rg areanalogous constructs of E-selectin, P-selectin and CD22β joined to ahuman G1 immnunoglobulin Fc domain (R. M. Nelson et al., 1993, supra; I.Stamenkovic et al., 1991, Cell 66, 1133-1144). Purified chimera wereprovided by A. Varki. Soluble P-selectin was purchased from R&D Systems.Protein A Sepharose 4 Fast Flow beads were purchased from PharmaciaBiotech. Anti-L-selectin monoclonal anitbodies: SK11 was obtained fromBecton-Dickinson, San Jose, Calif.; DREG-56, an L-selectin specificmonoclonal antibody, was purchased from Endogen, Cambridge, Mass. The 2'NH₂ modified CTP and UTP were prepared according to Pieken et. al.(1991, Science 253:314-317). DNA oligonucleotides were synthesized byOperon. All other reagents and chemicals were purchased from commercialsources. Unless otherwise indicated, experiments utilized HSMC buffer (1mM CaCl₂, 1 mM MgCl₂, 150 mM NaCl, 20.0 mM HEPES, pH 7.4).

B) SELEX

The SELEX procedure is described in detail in U.S. Pat. No. 5,270,163and elsewhere. The nucleotide sequence of the synthetic DNA template forthe LS-Rg SELEX was randomized at 40 positions. This variable region wasflanked by N7 5' and 3' fixed regions (40N7). 40N7 has the sequence 5'gggaggacgaugcgg-40N-cagacgacucgcccga 3' (SEQ ID NO: 64). All C and Uhave 2'NH₂ substituted for 2'OH on the ribose. The primers for the PCRwere the following:

N7 5' Primer 5' taatacgactcactatagggaggacgatgcgg 3' (SEQ ID NO: 65)

N7 3' Primer 5' tcgggcgagtcgtcctg 3' (SEQ ID NO: 66) The fixed regionsinclude primer annealing sites for PCR and cDNA synthesis as well as aconsensus T7 promoter to allow in vitro transcription. The initial RNApool was made by first Klenow extending 1 nmole of synthetic singlestranded DNA and then transcribing the resulting double strandedmolecules with T7 RNA polymerase. Klenow extension conditions: 3.5nmoles primer 5N7, 1.4 nmoles 40N7, 1X Klenow Buffer, 0.4 mM each ofdATP, dCTP, dGTP and dTTP in a reaction volume of 1 ml.

For subsequent rounds, eluted RNA was the template for AMV reversetranscriptase mediated synthesis of single-stranded cDNA. Thesesingle-stranded DNA molecules were converted into double-strandedtranscription templates by PCR amplification. PCR conditions were 50 mMKCl, 10 mM Tris-Ci, pH 8.3, 7.5 mM MgCl₂, 1 mM of each dATP, dCTP, dGTP,and dTTP, and 25 U/ml of Taq DNA polymerase. Transcription reactionscontained 0.5 mM DNA template, 200 nM T7 RNA polymerase, 80 mM HEPES (pH8.0), 12 mM MgCl₂, 5 mM DTT, 2 mM spermidine, 2 mM each of 2'OH ATP,2'OH GTP, 2'NH₂ CTP, 2'NH₂ UTP, and 250 nM α³² p 2'OH ATP.

The strategy for partitioning LS-Rg/RNA complexes from unbound RNA isoutlined in Tables VIIa and VIIb. First, the RNA pool was incubated withLS-Rg immobilized on protein A sepharose beads in HSMC buffer. Second,the unbound RINA was removed by extensive washing. Third, the RNAmolecules bound at the carbohydrate binding site were specificallyeluted by incubating the washed beads in HMSC buffer containing 5 mMEDTA in place of divalent cations. The 5 mM elution was followed by anon-specific 50 mM EDTA elution. LS-Rg was coupled to protein Asepharose beads according to the manufacturer's instructions (PharmaciaBiotech).

The 5 mM EDTA elution is a variation of a specific site elutionstrategy. Although it is not a priori as specific as elution bycarbohydrate competition, it is a general strategy for C-type (calciumdependent binding) lectins and is a practical alternative when the costand/or concentration of the required carbohydrate competitor isunreasonable (as is the case with sialyl-Lewis^(X)). This scheme isexpected to be fairly specific for ligands that form bonds with thelectin's bound Ca⁺⁺ because the low EDTA concentration does notappreciably increase the buffer's ionic strength and the conformation ofC-type lectins is only subtly altered in the absence of bound calcium(unpublished obervations cited by K. Drickamer, 1993, Biochem. Soc.Trans. 21:456-459).

In the initial SELEX rounds, which were performed at 4° C., the densityof immobilized LS-Rg was 16.7 pmoles/μl of Protein A Sepharose 4 FastFlow beads. In later rounds, the density of LS-Rg was reduced (TablesVIIa and VIIb), as needed, to increase the stringency of selection. Atthe seventh round, the SELEX was branched and continued in parallel at4° C. (Table VIIa) and at room temperature (Table VIIb). Wash andelution buffers were equilibrated to the relevant incubationtemperature. Beginning with the fifth round, SELEX was often done atmore than one LS-Rg density. In each branch, the eluted material fromonly one LS-Rg density was carried forward.

Before each round, RNA was batch adsorbed to 100 μl of protein Asepharose beads for 1 hour in a 2 ml siliconized column. Unbound RNA andRNA eluted with minimal washing (two volumes) were combined and used forSELEX input material. For SELEX, extensively washed, immobilized LS-Rgwas batch incubated with pre-adsorbed RNA for 1 to 2 hours in a 2 mlsiliconized column with constant rocking. Unbound RNA was removed byextensive batch washing (200 to 500 μl HSMC/wash). Bound RNA was elutedas two fractions; first, bound RNA was eluted by incubating and washingcolumns with 5 mM EDTA in HSMC without divalent cations; second, theremaining elutable RNA was removed by incubating and/or washing with 50mM EDTA in HSMC without divalents. The percentage of input RNA that waseluted is recorded in Tables VIIa and VIIb. In every round, an equalvolume of protein A sepharose beads without LS-Rg was treatedidentically to the SELEX beads to determine background binding. Allunadsorbed, wash and eluted fractions were counted in a Beckman LS6500scintillation counter in order to monitor each round of SELEX.

The eluted fractions were processed for use in the following round(Tables VIIa and VIIb). After extracting with phenol/chloroform andprecipitating with isopropanol/ethanol (1:1, v/v), the RNA was reversetranscribed into cDNA by AMV reverse transcriptase either 1) at 48° C.for 15 minutes and then 65° C. for 15 minutes or 2) at 37° C. and 48° C.for 15 minutes each, in 50 mM Tris-Cl pH (8.3), 60 mM NaCl, 6 mMMg(OAc)₂, 10 mM DTT, 100 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4unit/μl AMV RT. Transcripts of the PCR product were used to initiate thenext round of SELEX.

C) Nitrocellulose Filter Partitioning

As described in SELEX Patent Applications, the nitrocellulose filterpartitioning method was used to determine the affinity of RNA ligandsfor LS-Rg and for other proteins. Filter discs (nitrocellulose/celluloseacetate mixed matrix, 0.45 μm pore size, Millipore) were placed on avacuum manifold and washed with 2 ml of HSMC buffer under vacuum.Reaction mixtures, containing ³² p labeled RNA pools and unlabeledLS-Rg, were incubated in HSMC for 10-20 min at 4° C., room temperatureor 37° C., filtered, and then immediately washed with 4 ml HSMC at thesame temperature. The filters were air-dried and counted in a BeckmanLS6500 liquid scintillation counter without fluor.

LS-Rg is a dimeric protein that is the expression product of arecombinant gene constructed by fusing the DNA sequence that encodes theextracellular domains of human L-selectin to the DNA that encodes ahuman IgG₂ Fc region. For affinity calculations, we assume one RNAligand binding site per LS-Rg monomer (two per dimer). The monomerconcentration is defined as 2 times the LS-Rg dimer concentration. Theequilibrium dissociation constant, K_(d), for an RNA pool or specificligand that binds monophasically is given by the equation

    Kd= Pf! Rf!/ RP!

where,

Rf!=free RNA concentration

Pf!=free LS-Rg monomer concentration

RP!=concentration of RNA/LS-Rg complexes

KD =dissociation constant

A rearrangement of this equation, in which the fraction of RNA bound atequilibrium is expressed as a function of the total concentration of thereactants, was used to calculate Kds of monophasic binding curves:

    q=(P.sub.T +R.sub.T +K.sub.D -((P.sub.T +R.sub.T +K.sub.D).sup.2 -4 P.sub.T R.sub.T).sup.1/2)

q=fraction of RNA bound

P_(T) !=2×(total LS-Rg concentration)

R_(T) !32 total RNA concentration

Many ligands and evolved RNA pools yield biphasic binding curves.Biphasic binding can be described as the binding of two affinity speciesthat are not in equilibrium. Biphasic binding data were evaluated withthe equation

    q=2P.sub.t +R.sub.t +Kd.sub.1 +Kd.sub.2 - (P.sub.t +X.sub.1 R.sub.1 +K.sub.d1).sup.2 -4P.sub.t X.sub.1 R.sub.t !.sup.1/2

    - (P.sub.t +X.sub.2 R.sub.t +K.sub.d2).sup.2 -4P.sub.t X.sub.2 R.sub.t !1/2,

where X₁ and X₂ are the mole fractions of affinity species R₁ and R₂ andK_(d1) and K_(d2) are the corresponding dissociation constants. K_(D) swere determined by least square fitting K_(D) s were determined by leastsquare fitting of the data points using the graphics programKaleidagraph (Synergy Software, Reading , Pa.).

D) Cloning and Sequencing

Sixth, thirteenth (RT) and fourteenth (4° C.) round PCR products werere-amplified with primers which contain either a BamHI or a HinDIIIrestriction endonuclease recognition site. Using these restrictionsites, the DNA sequences were inserted directionally into the pUC9vector. These recombinant plasmids were transformed into E. coli strainDH5α (Life Technologies, Gaithersburg, Md.). Plasmid DNA was preparedaccording to the alkaline hydrolysis method (PERFECTprep, 5'-3',Boulder, Colo.). Approximately 150 clones were sequenced using theSequenase protocol (Amersham, Arlington Heights, Ill.). The resultingligand sequences are shown in Table VIII.

E) Cell Binding Studies

The ability of evolved ligand pools and cloned ligands to bind toL-selectin presented in the context of a cell surface was tested inexperiments with isolated human peripheral blood mononuclear cells(PBMCs). Whole blood, collected from normal volunteers, wasanticoagulated with 5 mM EDTA. Six milliliters of blood were layered ona 6 ml Histopaque gradient in 15 ml polyproylene tube and centrifuged(700 g) at room temperature for 30 minutes. The mononuclear cell layerwas collected, diluted in 10 ml of Ca⁺⁺ /Mg⁺⁺ -free DPBS (DPBS(-); Gibco14190-029) and centrifuged (225 g) for 10 minutes at room temperature.Cell pellets from two gradients were combined, resuspended in 10 ml ofDPBS(-) and recentrifuged as described above. These pellets wereresuspended in 100 μl of SMHCK buffer supplemented with 1% BSA. Cellswere counted in a hemocytometer, diluted to 2×10⁷ cells/ml in SMHCK/1%BSA and immediately added to binding assays. Cell viability wasmonitored by trypan blue exclusion.

For cell binding assays, a constant number of cells were titrated withincreasing concentrations of radiolabeled ligand. The test ligands wereserially diluted in DPBS(-)/1% BSA to 2-times the desired finalconcentration approximately 10 minutes before use. Equal volumes (25 μl)of each ligand dilution and the cell suspension (2×10⁷ cells/ml) wereadded to 0.65 ml eppendorf tubes, gently vortexed and incubated on icefor 30 minutes. At 15 minutes the tubes were revortexed. The ligand/PBMCsuspension was layered over 50 μl of ice cold phthalate oil(1:1=dinonyl:dibutyl phthalate) and microfuged (14,000 g) for 5 minutesat 4° C. Tubes were frozen in dry ice/ethanol, visible pellets amputatedinto scintillation vials and counted in Beckman LS6500 scintilationcounter as described in Example 7, paragraph C.

The specificity of binding to PBMCs was tested by competition with theL-selectin specific blocking monoclonal antibody, DREG-56, whilesaturability of binding was tested by competition with unlabeled RNA.Experimental procedure and conditions were like those for PBMC bindingexperiments, except that the radiolabeled RNA ligand (finalconcentration 5 nM ) was added to serial dilutions of the competitorbefore mixing with PBMCs.

G) Inhibition of Selectin Binding to sialyl-Lewis^(X)

The ability of evolved RNA pools or cloned ligands to inhibit thebinding of LS-Rg to sialyl-Lewis^(X) was tested in competive ELISAassays (C. Foxall et al., 1992, supra). For these assays, the wells ofCorning (25801) 96 well microtiter plates were coated with 100 ng of asialyl-Lewis^(X) /BSA conjugate, air dried overnight, washed with 300 μlof PBS(-) and then blocked with 1% BSA in SHMCK for 60 min at roomtemperature. RNA ligands were incubated with LS-Rg in SHMCK/1% BSA atroom temperature for 15 min. After removal of the blocking solution, 50μl of LS-Rg (10nM) or a LS-Rg (10nM )/RNA ligand mix was added to thecoated, blocked wells and incubated at room temperature for 60 minutes.The binding solution was removed, wells were washed with 300 μl ofPBS(-) and then probed with HRP conjugated anti-human IgG, at roomtemperature to quantitate LS-Rg binding. After a 30 minute incubation atroom temperature in the dark with OPD peroxidase substrate (SigmaP9187), the extent of LS-Rg binding and percent inhibition wasdetermined from the OD₄₅₀.

EXAMPLE 8 RNA Ligands to LS-Rg

A. SELEX

The starting RNA pool for SELEX, randomized 40N7 (SEQ ID NO: 63),contained approximately 10¹⁵ molecules (1 nmol RNA). The SELEX protocolis outlined in Tables VIIa and VIIb. The dissociation constant ofrandomized RNA to LS-Rg is estimated to be approximately 10 μM. Nodifference was observed in the RNA elution profiles with 5 mM EDTA fromSELEX and background beads for rounds 1 and 2, while the 50 mM elutionproduced a 2-3 fold excess over background (Table VIIa). The 50 mMeluted RNA from rounds 1 and 2 were amplified for the input material forrounds 2 and 3, respectively. Beginning in round 3, the 5 mM elutionfrom SELEX beads was significantly higher than background and wasprocessed for the next round's input RNA. The percentage of input RNAeluted by 5 mM EDTA increased from 0.5 in the first round to 8.4% inround 5 (Table VIIa). An additional increase in specifically eluted RNAfrom the 10 μM LS-Rg beads was not observed in round 6 (Table VIIa). Toincrease the stringency of selection, the density of immobilized LS-Rgwas reduced ten fold in round 5 with further reductions in proteindensity at later rounds. The affinity of the selected pools rapidlyincreased and the pools gradually evolved biphasic bindingcharacteristics.

Binding experiments with 6th round RNA revealed that the affinity of theevolving pool for L-selectin was temperature sensitive. Beginning withround 7, the SELEX was branched; one branch was continued at 4° C.(Table VIla) while the other was conducted at room temperature (TableVIIb). Bulk sequencing of 6th, 13th (rm temp) and 14th (4° C.) RNA poolsrevealed noticeable non-randomness at round six and dramaticnon-randomess at the later rounds. The 6th round RNA boundmonophasically at 4° C. with a dissociation constant of approximately 40nM, while the 13th and 14th round RNAs bound biphasically with highaffinity Kds of approximately 700 pM. The molar fraction of the twopools that bound with high affinity were 24% and 65%, repectively. Thebinding of all tested pools required divalent cations. In the absence ofdivalent cations, the Kds of the 13th and 14th round pools increased to45 nM and 480 nM , respectively (HSMC, minus Ca⁺⁺ /Mg⁺⁺, plus 2 mMEDTA).

To monitor the progress of SELEX, ligands were cloned and sequenced fromrounds 6, 13 (rm temp) and 14 (4° C.). Sequences were aligned manuallyand with the aid of a computer program that determines consensussequences from frequently occuring local alignments.

B. RNA Sequences

In Table VIII, ligand sequences are shown in standard single letter code(Cornish-Bowden, 1985 NAR 13: 3021-3030). The letter/number combinationbefore the "." in the ligand name indicates whether it was cloned fromthe round 6, 13 or 14 pools. Only the evolved random region is shown inTable VIII. Any portion of the fixed region is shown in lower caseletters. By definition, each clone includes both the evolved sequenceand the associated fixed region, unless specifically stated otherwise.From the sixth, thirteenth and fourteenth rounds, respectively, 26 of48, 8 of 24 and 9 of 70 sequenced ligands were unique. A unique sequenceis operationally defined as one that differs from all others by three ormore nucleotides. Sequences that were isolated more than once, areindicated by the parenthetical number, (n), following the ligand isolatenumber. These clones fall into thirteen sequence families (I-XIII) and agroup of unrelated sequences (Orphans)(SEQ ID NOs: 67-117).

Two families, I and III, are defined by ligands from multiple lineages.Both families occur frequently in round 6, but only one family IIIligand was identified in the final rounds. Six families (IV, V, VI, VII,VIII, and possibly II) are each defined by just two lineages whichlimits confidence in their consensus sequences. Five families (IXthrough XIII) are defined by a single lineage which precludesdetermination of consensus sequences.

Ligands from family II dominate the final rounds: 60/70 ligands in round14 and 9/24 in round 13. Family II is represented by three mutationalvariations of a single sequence. One explanation for the recovery of asingle lineage is that the ligand's information content is extremelyhigh and was therefore represented by a unique species in the startingpool. Family II ligands were not detected in the sixth round which isconsistent with a low frequency in the initial population. Analternative explanation is sampling error. Note that a sequence ofquestionable relationship was detected in the sixth round.

The best defined consensus sequences are those of family I, AUGUGUA (SEQID NO: 118), and of family III, AACAUGAAGUA (SEQ ID NO: 120), as shownin Table VIII. Family III has two additional, variably spaced sequences,AGUC and ARUUAG, that may be conserved. The tetranucleotide AUGW isfound in the consensus sequence of families I, III, and VII and infamilies II, VIII and IX. If this sequence is significant, it suggeststhat the conserved sequences of ligands of family VIII are circularlypermuted. The sequence AGAA is found in the consensus sequence offamilies IV and VI and in families X and XIII.

D. Affinities

The dissociation constants for representative ligands from rounds 13 and14, including all orphans, were determined by nitrocellulose filterbinding experiments and are listed in Table IX. These calculationsassume two RNA ligand binding sites per chimera. The affinity of randomRNA cannot be reliably determined but is estimated to be approximately10 μM.

In general, ligands bind monophasically with dissociation constantsranging from 50 pM to 15 nM at 4° C. Some of the highest affinityligands bind biphasically. Although ligands of families I, VII, X andorphan F14.70 bind about equally well at 4° C. and room temperature, ingeneral the affinities decrease with increasing temperature. Theobserved affinities substantiate the contention that it is possible toisolate oligonucleotide ligands with affinities that are serveral ordersof magnitude greater than that of carbohydrate ligands.

EXAMPLE 9 Specificity of RNA Ligands

The affinity of L-selectin ligands to ES-Rg, PS-Rg and CD22β-Rg weredetermined by nitrocellulose partitioning. As indicated in Table X, theligands are highly specific for L-selectin. In general, a ligand'saffinity for ES-Rg is 10³ -fold lower and that for PS-Rg is about 10⁴-fold less than for LS-Rg. Binding above background is not observed forCD22β-Rg at the highest protein concentration tested (660 nM ),indicating that ligands do not bind the Fc domain of the chimericconstructs nor do they have affinity for the sialic acid binding site ofan unrelated lectin. The specificity of oligonucleotide ligand bindingcontrasts sharply with the binding of cognate carbohydrates by theselectins and confirms the assertion that SELEX ligands will havegreater specificity than carbohydrate ligands.

EXAMPLE 10

Binding of RNA Ligands to PBMCs

Since the L-selectin ligands were isolated against purified, immobilizedprotein, it is essential to demonstrate that they bind L-selectinpresented in the context of a cell surface. Comparison of 2nd and 9thround RNAs (FIG. 2) shows that the evolved (9th round) ligand pool bindsisolated PBMCs with high affinity and, as expected for specific binding,in a saturable fashion. The binding of round 2 RNA appears to benon-saturable as is characteristic of non-specific binding. The clonedligand, F14.12 (SEQ ID NO: 78), also binds in a saturable fashion with adissociation constant of 1.3 nM , while random 40N7 (SEQ ID NO: 64)resembles round 2 RNA (FIG. 3). The saturability of binding is confrimedby the data in FIG. 4; >90% of 5 nM ³² P-labeled F14.12 RNA binding iscompeted by excess cold RNA. Specificity is demonstrated by the resultsin FIG. 5; binding of 5 nM ³² P-labeled F 14.12 RNA is completelycompeted by the anti-L-selectin blocking monoclonal antibody, DREG-56,but is unaffected by an isotype-matched irrelevant antibody. These datavalidate the feasibilty of using immobilized, purified protein toisolate ligands against a cell surface protein and the bindingspecificity of F 14.12 to L-selectin in the context of a cell surface.

EXAMPLE 11 Inhibition of Binding to sialy-Lewis^(X)

Oligonucleotide ligands, eluted by 2-5 mM EDTA, are expected to derivepart of their binding energy from contacts with the lectin domain'sbound Ca⁺⁺ and consequently, are expected to compete withsialyl-Lewis^(X) for binding. The ability of ligand F14.12 (SEQ ID NO:78) to inhibit LS-Rg binding to immobilized sialy-Lewis^(X) wasdetermined by competition ELISA assays. As expected, 4 mM EDTA reducedLS-Rg binding 7.4-fold, while 20 mM round 2 RNA did not inhibit LS-Rgbinding. Carbohydrate binding is known to be Ca⁺⁺ dependent; theaffinity of round 2 RNA is too low to bind 10 nM LSRg (Table VII).

In this assay F14.12 RNA inhibits LS-Rg binding in a concentrationdependent manner with an IC₅₀ of about 10 nM (FIG. 6). Completeinhibition is observed at 50 nM F14.12. The observed inhibition isreasonable under the experimental conditions; the Kd of F14.12 at roomtemperature is about 1 nM (Table IX) and 10 nM LS-Rg is 20 nM bindingsites. These data verify that RNA ligands compete with sialyl-Lewis^(X)for LS-Rg binding and support the contention that low concentrations ofEDTA specifically elute ligands that bind the lectin domain'scarbohydrate binding site.

EXAMPLE 12 Secondary Structure of High Affinity Ligands

In favorable instances, comparative analysis of aligned sequences allowsdeduction of secondary structure and structure-function relationships.If the nucleotides at two positions in a sequence covary according toWatson-Crick base pairing rules, then the nucleotides at these positionsare apt to be paired. Nonconserved sequences, especially those that varyin length are not apt to be directly involved in function, while highlyconserved sequence are likely to be directly involved.

Comparative analysis of the family I alignment suggests a hairpinstructure in which the consensus sequence, AUGUGUGA, is contained withina variable size loop (FIG. 7a). The stem sequences are not conserved andmay be either 5' or 3'-fixed or variable sequence. The one ligand thatdoes not form a stem, F14.25 (SEQ ID NO: 73), has a significantly loweraffinity than the other characterized ligands (Table IX).

The proposed structure for family III is also a hairpin with theconserved sequence, AACAUGAAGUA, contained within a variable length loop(FIG. 7b). The 5'-half of the stem is 5'-fixed sequence which mayaccount in part for the less highly conserved sequence, AGUC.

Although there is no alignment data for family II, the sequence foldsinto a pseudoknot (FIG. 7c). Three attractive features of this modelare 1) the helices stack on one another, 2) the structure utilizes onlyvariable sequence and 3) the structure is compatible with the majorvariant sequences.

EXAMPLE 13 ssDNA Ligands to Human L-Selectin

Experimental Procedures

A) Materials

Unless otherwise indicated, all materials used in the ssDNA SELEXagainst the L-selectin/IgG2 chimera, LS-Rg, were identical to those ofExample 7. The buffer for SELEX experiments was 1 mM CaCl₂, 1 mM MgCl₂,100 mM NaCl, 10.0 mM HEPES, pH 7.4. The buffer for all binding affinityexperiments differed from the above in containing 125 mM NaCl, 5 miMKCI, and 20 mM HEPES, pH 7.4.

B) SELEX

The SELEX procedure is described in detail in U.S. Pat. No. 5,270,163and elsewhere. The nucleotide sequence of the synthetic DNA template forthe LS-Rg SELEX was randomized at 40 positions. This variable region wasflanked by BH 5' and 3' fixed regions. The random DNA template wastermed 40BH (SEQ ID NO: 126) and had the following sequence:5'-ctacctacgatctgactagc<40N>gcttactctcatgtagttcc-3'. The primers for thePCR were the following: 5' Primer: 5'-ctacctacgatctgactagc-3' (SEQ IDNO: 127) and 3' Primer: 5'-ajajaggaactacatgagagtaagc-3'; j=biotin (SEQID NO: 128). The fixed regions include primer annealing sites for PCRamplification. The initial DNA pool contained 500 pmoles of each of twotypes of single-stranded DNA: 1) synthetic ssDNA and 2) PCR amplified,ssDNA from 1 nmole of synthetic ssDNA template.

For subsequent rounds, eluted DNA was the template for PCRamplification. PCR conditions were 50 mM KCI, 10 mM Tris-Cl, pH 8.3, 7.5mM MgCl₂, 1 mM of each dATP, dCTP, dGTP, and dTTP and 25 U/ml of theStoffel fragment of Taq DNA polymerase. After PCR amplification, doublestranded DNAs were end-labeled using γ³² P-ATP. Complementary strandswere separated by electrophoresis through an 8% polyacrylamide/7M ureagel. Strand separation results from the molecular weight difference ofthe strands due to biotintylation of the 3' PCR primer. In the finalrounds, DNA strands were separated prior to end labelling in order toachieve high specific activity. Eluted fractions were processed byethanol precipitation.

The strategy for partitioning LS-Rg/RNA complexes from unbound RNA wasas described in Example 7, paragraph B, except that 2 mM EDTA wasutilized for specific elution. The SELEX strategy is outlined in TableXI.

C) Nitrocellulose Filter Partitioning

As described in SELEX Patent Applications and in Example 7, paragraph C,the nitrocellulose filter partitioning method was used to determine theaffinity of RNA ligands for LS-Rg and for other proteins. For theseexperiments a Gibco BRL 96 well manifold was substituted for the 12 wellMillipore manifold used in Example 7 and radioactivity was determinedwith a Fujix BAS 100 phosphorimager. Binding data were analysed asdescribed in Example 7, paragraph C.

D) Cloning and Sequencing

Thirteenth and fifteenth round PCR products were re-amplified withprimers which contain either a BamHI or a HinDIII restrictionendonuclease recognition site. Approximately 140 ligands were cloned andsequenced using the procedures described in Example 7, paragraph D. Theresulting sequences are shown in Table XII.

E) Cell Binding Studies

The ability of evolved ligand pools to bind to L-selectin presented inthe context of a cell surface was tested in experiments with isolatedhuman peripheral blood mononuclear cells (PBMCs) as described in Example7, paragraph E.

EXAMPLE 14 ssDNA Ligands to L-Selectin

A. SELEX

The starting ssDNA pool for SELEX, randomized 40BH (SEQ ID NO: 126),contained approximately 10¹⁵ molecules (1 nmol ssDNA). The dissociationconstant of randomized ssDNA to LS-Rg is estimated to be approximately10 μM. The SELEX protocol is outlined in Table XI.

The initial round of SELEX was performed at 4° C. with an LS-Rg densityof 16.7 pmole/μl of protein A sepharose beads. Subsequent rounds were atroom temperature except as noted in Table XI. The 2 mM EDTA elution wasomitted from rounds 1-3. The signal to noise ratio of the 50 mM EDTAelution in these three rounds was 50, 12 and 25, respectively (TableXI). These DNAs were amplified for the input materials of rounds 2-4.Beginning with round 4, a 2 mM EDTA elution was added to the protocol.In this and all subsequent rounds, the 2 mM EDTA eluted DNA wasamplified for the next round's input material.

To increase the stringency of selection, the density of immobilizedLS-Rg was reduced ten fold in round 4 with further reductions in proteindensity at rounds 9, 11, 14 and 15 (Table XI). Under these conditions arapid increase in the affinity of the selected pools was observed(Tables XI); at 4° C., the dissociation constant of round 7 ssDNA was 60nM .

Binding experiments with 7th round DNA revealed that the affinity of theevolving pool for L-selectin was weakly temperature sensitive (Kds: 60nM, 94 nM and 230 nM at 4° C., room temperature and 37° C.,respectively). To enhance the selection of ligands that bind atphysiological temperature, rounds 8 and 13 were performed at 37° C.Although still temperature sensitive, the affinity of round 15 ssDNA wasoptimal at room temperature (160 pM), with 3-fold higher Kds at 4° C.and 37° C.

Bulk sequencing of DNA pools indicates some non-randomness at round 5and dramatic non-randomness at round 13. Ligands were cloned andsequenced from rounds 13 and 15. Sequences were aligned mannually andwith the aid of a Nexstar computer program that determines consensussequences from frequently occuring local alignments.

B. ssDNA Sequences

In Table XII, ligand sequences are shown in standard single letter code(Cornish-Bowden, 1985 NAR 13: 3021-3030). Only the evolved random regionis shown in Table XII. Any portion of the fixed region is shown in lowercase letters. By definition, each clone includes both the evolvedsequence and the associated fixed region, unless specifically statedotherwise. Thirty-nine of sixty seven sequenced ligands were unique. Aunique sequence is operationally defined as one that differs from allothers by three or more nucleotides. Sequences that were isolated morethan once are indicated by the parenthetical number, (n), following theligand isolate number. These clones fall into six families and a groupof unrelated sequences or orphans (Table XII)(SEQ ID NOs: 129-167).

Family 1 is defined by 52 ligands from 28 lineages and has a welldefined consensus sequence, TACAAGGYGYTAVACGTA (SEQ ID NO: 168). Theconservation of the CAAGG and ACG and their 6 nucleotide spacing isnearly absolute (Table XII). The consensus sequence is flanked byvariable but complementary sequences that are 3 to 5 nucleotides inlength. The statistical dominance of family 1 suggests that theproperties of the bulk population are a reflection of those of family 1ligands. We note that ssDNA family 1 and 2' NH2 family 1 share a commonsequence, CAAGGCG and CAAGGYG, respectively.

Family 2 is respresented by a single sequence and is related tofamily 1. The ligand contains the absolutely conserved CAAGG and highlyconserved ACG of family 1 although the spacing between the two elementsis strikingly different (23 compared to 6 nucleotides).

Families 4-6 are each defined by a small number of ligands which limitsconfidence in their consensus sequence, while family 7 is defined by asingle sequence which precludes determination of a consensus. Family 5appears to contain two conserved sequences, AGGGT and RCACGAYACA, thepositions of which are circularly permuted.

D. Affinities

The dissociation constants of representative ligands from Table XII areshown in Table XIII. These calculations assume two ssDNA ligand bindingsites per chimera. The affinity of random ssDNA cannot be reliablydetermined but is estimated to be approximately 10 μM.

At room temperature, the dissociation constants range from 43 pM to 1.8nM which is at least a 5×10³ to 2×10⁵ fold improvement over randomizedssDNA (Table XIII). At 37° C., the Kds range from 130 pM to 23 nM. Theextent of temperature sensitivity varies from insensitive (ligands 122and 127 (SEQ ID NO: 159 and 162)) to 80-fold (ligand 112 (SEQ ID NO:135)). In general, among family 1 ligands the affinity of those fromround 15 is greater than that of those from round 13. For the bestligands (208, 227, 230 and 233 (SEQ ID NOS: 133, 134, 132, and 146)),the difference in affinity at room temperature and 37° C. is about4-fold.

The observed affinities of the evolved ssDNA ligand pools reaffirm ourcontention that it is possible to isolate oligonucleotide ligands withaffinities that are several orders of magnitude greater than that ofcarbohydrate ligands.

EXAMPLE 15 Specificity of ssDNA Ligands

The affinity of representative cloned ligands for LS-Rg, ES-Rg, PS-Rg,CD22βRg and WGA was determined by nitrocellulose partitioning and theresults shown in Table XIV. The ligands are highly specific forL-selectin. The affinity for ES-Rg is about 10³ -fold lower and that forPS-Rg is about 5×10³ -fold less than for LS-Rg. Binding above backgroundis not observed for CD22β-Rg or for WGA at 0.7 and 1.4 μM protein,respectively, indicating that ligands neither bind the Fc domain of thechimeric constructs nor have affinity for unrelated sialic acid bindingsites.

The specificity of oligonucleotide ligand binding contrasts sharply withthe binding of cognate carbohydrates by the selectins and reconfirms theassertion that SELEX ligands will have greater specificity thancarbohydrate ligands.

EXAMPLE 16 Cell Binding Studies

Round 15 ssDNA pool was tested for its ability to bind to L-selectinpresented in the context of a peripheral blood mononuclear cell surfaceas described in Example 7, paragraph E. The evolved pool was tested bothfor affinity and for specificity by competition with an anti-L-selectinmonoclonal antibody. FIG. 8 shows that the round 15 ssDNA pool bindsisolated PBMCs with a dissociation constant of approximately 1.6 nM and,as is expected for specific binding, in a saturable fashion. FIG. 9directly demonstrates specificity of binding; in this experiment,binding of 2 nM ³² P-labeled round 15 ssDNA is completely competed bythe anti-L-selectin blocking monoclonal antibody, DREG-56, but isunaffected by an isotype-matched irrelevant antibody.

These data validate the feasibilty of using immobilized, purifiedprotein to isolate ligands against a cell surface protein and thebinding specificity of round 15 ssDNA ligands to L-selectin in thecontext of a cell surface.

EXAMPLE 17 Secondary Structure of High Affinity Ligands

In favorable instances, comparative analysis of aligned sequences allowsdeduction of secondary structure and structure-function relationships.If the nucleotides at two positions in a sequence covary according toWatson-Crick base pairing rules, then the nucleotides at these positionsare apt to be paired. Nonconserved sequences, especially those that varyin length are not apt to be directly involved in function, while highlyconserved sequence are likely to be directly involved.

Comparative analysis of the family 1 alignment suggests a hairpinsecondary structure which is shown in FIG. 10. In the figure, concensusnucleotides are specified, with invariant nucleotides in bold type. Tothe right of the stem is a matrix showing the number of occurances ofparticular base pairs for the position in the stem that is on the sameline. The matrix illustrates that stem is validated by Watson-Crickcovariation. For example, the nucleotide pair on the second line of thematrix is always a Watson-Crick pair (22/22) and is usually an AT(17/22). The stems are 6 or 7 base pairs in length (FIG. 10); the stemposition corresponding to line seven is base paired in 12/22 ligandswhile that corresponding to line eight of the matrix is paired in onlyone ligand indicating the end of the stem.

There are two major stem variants. In one group of twenty ligands (204through 227 (SEQ ID NOS: 129-134), Table XII), from 6 lineages, thefourth position from the loop is a TA base pair. The stem of a secondgroup of 19 ligands (112 through 233 (SEQ ID NOS: 135-146), Table XII),from 12 lineages, has a GA mismatch at the fourth position. In bothgroups, the base pair at the second and third positions from the loopare AT and TA, respectively (FIG. 10).

Twelve nucleotides of the consensus sequence, AAGGYGYTAVAC (SEQ ID NO:168), are contained in the loop which is closed by the invariant C and G(FIG. 10). Although interactions of the loop nucleotides are not definedby the data, the invaiant T and adjacent Y may interact with theinvariant AG.

Initial experiments to deduce the minimal sequence of family 1 ligandsthat is necessary for high affinity binding indicate that more than thehairpin is required. The affinity of a 41 nucleotide truncate of ligand201 (SEQ ID NO: 173) is reduced about 150-fold compared to the fulllength ligand, but the affinity of a 49-mer (SEQ ID NO: 172) is notsignigicantly altered (Table XII and XIII).

EXAMPLE 18 2'NH₂ RNA Ligands to Human E-Selectin

Experimental Procedures

A) Materials

ES-Rg is a chimeric protein in which the extracellular domain of humanE-selectin is joined to the Fc domain of a human GI immunoglobulin (R.M. Nelson et al., 1993, supra). Purified chimera were provided by A.Varki. Unless otherwise indicated, all materials used in this SELEX aresimilar to those of Examples 7 and 13.

B) SELEX

The SELEX procedure is described in detail in U.S. Pat. No. 5,270,163and elsewhere. The rationale and experimental procedures are the same asthose described in Examples 7 and 13.

EXAMPLE 19 ssDNA Ligands to Human P-Selectin

Experimental Procedures

A) Materials PS-Rg is a chimeric protein in which the lectin, EGF, andthe first two CRD domains of human P-selectin are joined to the Fcdomain of a human G1 immunoglobulin (R. M. Nelson et al., 1993, supra).Purified chimera is provided by A.Varki. Soluble P-selectin is purchasedfrom R&D Systems. Unless otherwise indicated, all materials used in thessDNA SELEX against the P-selectin/IgG₁ chimera, PS-Rg, are identical tothose of Examples 7 and 13.

B) SELEX

The SELEX procedure is described in detail in U.S. Pat. No. 5,270,163.The specific strategies and procedures for evolving high affinity ssDNAantagonists to P-selectin are described in Examples 7 and 13. Thesimilarity of L- and P-selectin's binding properties assures the successof the experimental procedures in isolating high affinity antagonists toP-selectin.

                                      TABLE I                                     __________________________________________________________________________    Wheat Germ Agglutinin Selex                                                       Total Protein                                                                       Total RNA                                                                           Gel Volume                                                                          Total Volume           Kd                               Round                                                                             (pmole)                                                                             (pmole)                                                                             (ul)  (ul)   % RNA Eluted                                                                         % RNA Amplified                                                                        (nM)                             __________________________________________________________________________     1  5,800 2,020 50    276    0.05   0.05     6,000,000                         2  5,800 1,070 50    276    0.12   0.12                                       3  5,800 1,770 50    280    0.21   0.21                                       4  5,800   900 50    263    3      3                                          5  5,800   500 50    271    28.5   28.5     600                               6a 5,800 1,000 50    282    28.8                                              6b   580 1,000 5     237    5.7    0.18     400                               7    580   940 5     245    12.8   0.87     320                               8    580   192 5     265    21.4   0.64     260                               9    58    170 0.5   215    3.8    0.06     130                              10    58    184 0.5   210    5.2    0.12     94                               11    58    180 0.5   210    2.3    0.07     68                               __________________________________________________________________________     Wheat Germ Lectin Sepharose 6 MB, WGA density, approximately 5 mg/ml of       gel or 116 uM.                                                                RNA Loading Conditions: Rounds 1-5, 2 hrs @ room temperature on roller;       incubation time reduced to 1 hr. for Rounds 6-11.                             RNA Elution Conditions: Rounds 1-5, 200 ul of 2 mM (GlcNAc)3, 15 min. @       room temperature on roller; 2x 200 ul wash with same buffer. Rounds 6: 20     ul of 0.2 mM (GlcNAc)3, incubated as above; washed sequentially with 200      ul of 0.5, 1, 1.5, 2 and 10 mM (GlcNAc)3. Rounds 7-8: 200 ul of 0.2 mM        (GlcNAc)3, incubated as in round 6; wash twice with same buffer; washed       sequentially with 3x 200 ul each, of 0.5, 1.0, 1.5, 2.0 and 10 mM             (GlcNAc)3. Rounds 9-11: incubated 15 @ room temperature in 200 ul of 1 mM     (GlcNAc); washed 2x with 200 ul of same buffer; incubation and washes         repeated with 1.5, 2.0 and 10 mM (GlcNAc).                                    % RNA Eluted: percentage of input RNA eluted with (GlcNAc)3                   % RNA Amplified: percentage of input RNA amplified; Rounds 1-5: entire        eluted RNA sample amplified. Rounds 6-11: pooled 2mM and 10 mM RNA,           amplified for subsequent round. Rounds 9-11: 1.5 mM RNA amplified             separately.                                                              

    TABLE II                                                                         - Sequence Alignment Wheat Germ Agglutinin                                     Ligand SEQ ID NO:                                                                FAMILY 1                                                                     11.8 4                                                                         ##STR1##                                                                       11.4(3) 5                                                                      ##STR2##                                                                       11.10 6                                                                        ##STR3##                                                                       11.35 7                                                                        ##STR4##                                                                       11.5 8                                                                         ##STR5##                                                                       11.26 9                                                                        ##STR6##                                                                       11.19 10                                                                       ##STR7##                                                                       11.15 11                                                                       ##STR8##                                                                       11.34 12                                                                       ##STR9##                                                                       6.8 13                                                                         ##STR10##                                                                      6.9 14                                                                         ##STR11##                                                                      6.23 15                                                                        ##STR12##                                                                      6.26 16                                                                        ##STR13##                                                                      6.14 17                                                                        ##STR14##                                                                      CONSENSUS: 56                                                                  ##STR15##                                                                        FAMILY 2                                                                     11.12 18                                                                       ##STR16##                                                                      11.24(2) 19                                                                    ##STR17##                                                                      11.27(2) 20                                                                    ##STR18##                                                                      11.32 21                                                                       ##STR19##                                                                      11.6 22                                                                        ##STR20##                                                                      CONSENSUS: 57                                                                  ##STR21##                                                                        FAMILY 3                                                                     11.13 23                                                                       ##STR22##                                                                      11.23 24                                                                       ##STR23##                                                                      6.3 25                                                                         ##STR24##                                                                      6.7 26                                                                         ##STR25##                                                                      6.20 27                                                                        ##STR26##                                                                      6.34 28                                                                        ##STR27##                                                                      6.35 29                                                                        ##STR28##                                                                      6.5 30                                                                         ##STR29##                                                                      6.16 31                                                                        ##STR30##                                                                      6.19 32                                                                        ##STR31##                                                                      6.21 33                                                                        ##STR32##                                                                      6.25 34                                                                        ##STR33##                                                                      6.33 35                                                                        ##STR34##                                                                      CONSENSUS: 58                                                                  ##STR35##                                                                        FAMILY 4                                                                     11.2 36                                                                        ##STR36##                                                                      11.33 37                                                                       ##STR37##                                                                      11.28 38                                                                       ##STR38##                                                                      11.7(4) 39                                                                     ##STR39##                                                                      CONSENSUS: 59                                                                  ##STR40##                                                                        FAMILY 5                                                                     11.20(5) 40                                                                    ##STR41##                                                                        FAMILY 6                                                                     6.15 41                                                                        ##STR42##                                                                      6.28 42                                                                        ##STR43##                                                                      CONSENSUS: 60                                                                  ##STR44##                                                                      6.24 43                                                                        ##STR45##                                                                      6.27 44                                                                        ##STR46##                                                                      11.3 45                                                                        ##STR47##                                                                      CONSENSUS: 61                                                                  ##STR48##                                                                        FAMILY 8                                                                     6.2 46                                                                         ##STR49##                                                                      6.6 (2) 47                                                                     ##STR50##                                                                      CONSENSUS: 62                                                                  ##STR51##                                                                        FAMILY 9                                                                     6.11 48                                                                        ##STR52##                                                                      6.13 49                                                                        ##STR53##                                                                      6.17 50                                                                        ##STR54##                                                                      CONSENSUS: 63                                                                  ##STR55##                                                                        ORPHANS                                                                      6.1 51                                                                         ##STR56##                                                                      6.4 52                                                                         ##STR57##                                                                      6.18 53                                                                        ##STR58##                                                                      11.30 54                                                                       ##STR59##                                                                      11.29 55                                                                       ##STR60##                                                                

                  TABLE III                                                       ______________________________________                                        Dissociation Constants of RNA Ligands to WGA                                  Ligand        SEQ ID NO:                                                                              Kd                                                    ______________________________________                                        Family 1                                                                      11.8           4        9.2        nM                                         11.4           5        32         nM                                         11.35          7        90         nM                                         11.5           8        44         nM                                         11.26          9        38         nM                                         11.19         10        22         nM                                         11.15         11        54         nM                                         11.34         12        92         nM                                         6.8           13        11         nM                                         6.9           14        396        nM                                         6.23          15        824        nM                                         6.14          17        <5%                                                   Family 2                                                                      11.12         18        15.2       nM                                         11.24         19        19.4       nM                                         11.27         20        30         nM                                         11.32         21        274        nM                                         11.6          22        702        nM                                         Family 3                                                                      11.13         23        <5%                                                   11.23         24        <5%                                                   6.3           25        120        nM                                         6.2           27        <5%                                                   6.34          28        <5%                                                   6.35          29        <5%                                                   6.5           30        678        nM                                         6.16          31        <5%                                                   6.19          32        74         nM                                         Family 4                                                                      11.2          36        62         nM                                         11.33         37        <5%                                                   11.28         38        9.2        nM                                         11.7          39        16         nM                                         Family 5                                                                      11.2          40        1.4        nM                                         Family 7                                                                      6.27          44        56         nM                                         11.3          45        410        nM                                         Family 8                                                                      6.6           47        <5%                                                   Family 9                                                                      6.11          48        <5%                                                   Orphans                                                                       11.3          54        56         nM                                         11.29         55        32         nM                                         ______________________________________                                         The Kds of ligands that show <5% binding at 1 uM WGA is estimated to be       >20 uM.                                                                  

                  TABLE IV                                                        ______________________________________                                        Specificity of RNA Ligands to WGA                                                    Kds for N-actyl-glucosamine Binding Lectins                                     Ligand 6.8  Ligand 11.20                                                                              Ligand 11.24                                 LECTIN   (SEQ ID NO: 13)                                                                           (SEQ ID NO: 40)                                                                           (SEQ ID NO: 19)                              ______________________________________                                        Triticum 11.4 nM     1.4 nM      19.2 nM                                      vulgare (WGA)                                                                 Canavalia                                                                              <5%*        <5%*        <5%*                                         ensiformis                                                                    (Con A)**                                                                     Datura   <5%*        11.2 μM  <5%*                                         stramonium                                                                    Ulex europaeus                                                                          4.4 μM  2.2 μM   <5%*                                         (UEA-II)                                                                      ______________________________________                                         *Less than 5% binding at 1 μM protein; estimated Kd >20 μM              **succinylated Con A                                                     

                  TABLE V                                                         ______________________________________                                        INHIBITION OF RNA LIGAND BINDING TO WHEAT GERM                                AGGULTININ                                                                    Ligand                                                                              SEQ ID NO:                                                                              Competitor                                                                              IC.sub.50 (μM)                                                                    Max Inhib                                                                            K.sub.c (μM)                       ______________________________________                                        6.8   13        (GlcNAc).sub.3                                                                           95    >95%   10.9                                  11.20 40        (GlcNAc).sub.3                                                                          120    >95%   8.4                                   11.24 19        (GlcNAc).sub.3                                                                          120    >95%   19.4                                  ______________________________________                                         K.sub.c is the dissociation constant of (GlcNAc).sub.3 calculated from        these data, assuming competitive inhibition and two RNA ligand binding        sites per dimer.                                                         

                  TABLE VI                                                        ______________________________________                                        INHIBITION OF WGA MEDIATED AGGLUTINATION                                      OF SHEEP ERYTHROCYTES                                                                 Inhibitory Concentration (μM)                                      Inhibitor SEQ ID NO:    Complete Partial                                      ______________________________________                                         6.8      13            0.5      0.12                                         11.20     40            0.5      0.12                                         11.24     19            *        2                                            (GlcNAc).sub.3          8        2                                            GlcNAc                  780      200                                          ______________________________________                                         *Complete inhibition of agglutination by ligand 11.24 was not observed in     this experiment.                                                         

                                      TABLE VIIa                                  __________________________________________________________________________    L-Selectin SELEX: 2'NH.sub.2 RNA at 4° C.                                                         % 5 mM                                                                             % 50 mM                                            Total                                                                             Total             EDTA EDTA                                          SELEX                                                                              RNA Protein                                                                           RNA:LS-                                                                            Bead                                                                              Total                                                                              Eluted                                                                             Eluted                                        Round #                                                                            pmoles                                                                            pmoles                                                                            Rg Ratio                                                                           Volume                                                                            Volume                                                                             RNA  RNA  Kd (nM)                                  __________________________________________________________________________    Rnd 0                                10,000                                   Rnd 1                                                                              1060                                                                              167.0                                                                             6.3  10 μL                                                                          ˜100 μL                                                                   0.498                                                                              0.301                                         Rnd 2                                                                              962 167.0                                                                             5.8  10 μL                                                                          ˜100 μL                                                                   0.306                                                                              0.114                                         Rnd 3                                                                              509 167.0                                                                             3.0  10 μL                                                                          ˜100 μL                                                                   1.480                                                                              0.713                                         Rnd 4                                                                              407 167.0                                                                             2.4  10 μL                                                                          ˜100 μL                                                                   5.010                                                                              1.596                                                                              434                                      Rnd 5                                                                              429 167.0                                                                             2.6  10 μL                                                                          ˜100 μL                                                                   8.357                                                                              7.047                                              439 16.7                                                                              26.3 10 μL                                                                          ˜100 μL                                                                   0.984                                                                              0.492                                                                              133                                      Rnd 6                                                                              452 167.0                                                                             2.7  10 μL                                                                          ˜100 μL                                                                   7.409                                                                              6.579                                              46  16.7                                                                              2.8  10 μL                                                                          ˜100 μL                                                                   3.468                                                                              1.312                                                                              37                                       Rnd 7                                                                              43  16.7                                                                              2.6  10 μL                                                                          ˜100 μL                                                                   8.679                                                                              2.430                                              44  16.7                                                                              2.6  10 μL                                                                          ˜100 μL                                                                   7.539                                                                              2.358                                              22  4.2 5.2  10 μL                                                                          ˜100 μL                                                                   2.748                                                                              1.298                                         Rnd 8                                                                              43  16.7                                                                              2.6  10 μL                                                                          ˜100 μL                                                                   8.139                                                                              1.393                                                                              33                                            23  4.2 5.5  10 μL                                                                          ˜100 μL                                                                   2.754                                                                              0.516                                         Rnd 9                                                                              23  4.2 5.5  10 μL                                                                          ˜100 μL                                                                   4.352                                                                              0.761                                         Rnd 10                                                                             21  4.2 5.0  10 μL                                                                          ˜100 μL                                                                   6.820                                                                              1.123                                                                              13                                            23  8.4 2.7  50 μL                                                                          ˜150 μL                                                                   14.756                                                                             1.934                                         Rnd 11                                                                             30  10.5                                                                              2.9  250 μL                                                                         ˜500 μL                                                                   0.707                                                                              0.033                                         Rnd 12                                                                             12  10.5                                                                              1.1  250 μL                                                                         ˜500 μL                                                                   3.283                                                                              0.137                                         Rnd 13                                                                             7   1   7    250 μL                                                                         ˜500 μL                                                                   4.188                                                                              0.136                                                                              0.3                                      Rnd 14                                                                             9   1   9    250 μL                                                                         ˜500 μL                                                                   4.817                                                                              0.438                                                                              0.7                                      __________________________________________________________________________     L-Selectin Rg was immobilized on Protein A Sepharose 4 Fast Flow. Protein     A density is approximately 6 mg/ml drained gel (143 uM).                      RNA Loading Conditions: All selections were carried out in the cold room.     The RNA used in each selection was first incubated for 30 minutes with 10     uL Protein A Sepharose in the cold room on a roller. Only RNA which flowe     through this column was used on the LSRg selection column. The RNA was        incubated on the selection column for 90 minutes on a roller before being     washed extensively with binding buffer (20 mM HEPES pH 7.4 150 mM NaCl, 1     mM MgCl.sub.2, 1 mM CaCl.sub.2.)                                              RNA Elution Conditions: RNA was eluted by incubating the extensivelywashe     columns in 100 uL of HEPES buffered EDTA (pH 7.4) for 30 minutes on a         roller followed by three 100 uL HEPES buffered EDTA washes.              

                                      TABLE VIIb                                  __________________________________________________________________________    L-Selectin SELEX: 2'NH.sub.2 RNA at 4° C.                                                         % 5 mM                                                                             % 50 mM                                            Total                                                                             Total             EDTA EDTA                                          SELEX                                                                              RNA Protein                                                                           RNA:LS-                                                                            Bead                                                                              Total                                                                              Eluted                                                                             Eluted                                        Round #                                                                            pmoles                                                                            pmoles                                                                            Rg Ratio                                                                           Volume                                                                            Volume                                                                             RNA  RNA  Kd (nM)                                  __________________________________________________________________________    Rnd 7                                                                              43  10.0                                                                              4.3  10 μL                                                                          ˜100 μL                                                                   1.205                                                                              0.463                                         Rnd 8                                                                              35  10  3.5  10 μL                                                                          ˜100 μL                                                                   6.642                                                                              0.401                                              35  10  3.5  10 μL                                                                          ˜100 μL                                                                   5.540                                                                              0.391                                         Rnd 9                                                                              24  2.5 9.6  10 μL                                                                          ˜100 μL                                                                   1.473                                                                              0.383                                                                              13                                       Rnd 10                                                                             30  6.3 4.9  250 μL                                                                         ˜500 μL                                                                   0.707                                                                              0.033                                         Rnd 11                                                                             12  6.3 1.9  250 μL                                                                         ˜500 μL                                                                   3.283                                                                              0.134                                         Rnd 12                                                                             6   0.6 9.4  250 μL                                                                         ˜500 μL                                                                   0.877                                                                              0.109                                                                              0.3                                      Rnd 13                                                                             1   0.6 1.4  250 μL                                                                         ˜500 μL                                                                   5.496                                                                              0.739                                                                              0.7                                      __________________________________________________________________________     L-Selectin Rg was immobilized on Protein A Sepharose 4 Fast Flow. Protein     A density is approximately 6 mg/ml drained gel (143 μM).                   RNA Loading Conditions: Selections were carried out at room temperature.      The RNA used in each selection was first incubated for 30 minutes with 10     uL Protein A Sepharose at room temp. Only RNA which flowed through this       column was used on the LSRg selection column. The RNA was incubated on th     selection column for 90 minutes on a roller before being washed               extensively with binding buffer (20 mM HEPES pH 7.4 150 mM NaCl, 1 mM         MgCl.sub.2, 1 mM CaCl.sub.2.)                                                 RNA Elution Conditions: RNA was eluted by incubating the extensivelywashe     columns in 100 μL of HEPES buffered EDTA (pH 7.4) for 30 minutes on a      roller followed by three 100 μL HEPES buffered EDTA washes.           

    TABLE VIII                                                                       - L-Selectin 2'NH.sub.2 RNA SELEX:                                             Ligand Sequences                                                               Ligand SEQ ID NO:                                                                Family I                                                                     F13.32(5) 67                                                                   ##STR61##                                                                      6.60(2) 68                                                                     ##STR62##                                                                      6.50(3) 69                                                                     ##STR63##                                                                      6.79 70                                                                        ##STR64##                                                                      F14.9 71                                                                       ##STR65##                                                                      F14.21 72                                                                      ##STR66##                                                                      F14.25 73                                                                      ##STR67##                                                                      F13.48(2) 74                                                                   ##STR68##                                                                      6.71 75                                                                        ##STR69##                                                                      6.28 76                                                                        ##STR70##                                                                      CONSENSUS: 118                                                                 ##STR71##                                                                        Family II                                                                    F14.20(26) 77                                                                  ##STR72##                                                                      F14.12(22) 78                                                                  ##STR73##                                                                      F14.11(12) 79                                                                  ##STR74##                                                                      F13.45(9) 80                                                                   ##STR75##                                                                      6.80 81                                                                        ##STR76##                                                                      CONSENSUS: 119                                                                 ##STR77##                                                                        Family III                                                                   6.17 82                                                                        ##STR78##                                                                      6.49 83                                                                        ##STR79##                                                                      6.16 84                                                                        ##STR80##                                                                      6.37 85                                                                        ##STR81##                                                                      6.78 86                                                                        ##STR82##                                                                      6.26 87                                                                        ##STR83##                                                                      6.40 88                                                                        ##STR84##                                                                      F13.57 89                                                                      ##STR85##                                                                      CONSENSUS: 120                                                                 ##STR86##                                                                        Family IV                                                                    6.58 90                                                                        ##STR87##                                                                      6.69 91                                                                        ##STR88##                                                                      CONSENSUS: 121                                                                 ##STR89##                                                                        Family V                                                                     6.56 92                                                                        ##STR90##                                                                      F13.55 93                                                                      ##STR91##                                                                      CONSENSUS: 122                                                                 ##STR92##                                                                        Family VI                                                                    F14.27 94                                                                      ##STR93##                                                                      F14.53 95                                                                      ##STR94##                                                                      CONSENSUS: 123                                                                 ##STR95##                                                                        Family VII                                                                   F13.42 96                                                                      ##STR96##                                                                      F13.51 97                                                                      ##STR97##                                                                      CONSENSUS: 124                                                                 ##STR98##                                                                        Family VIII                                                                  6.33(11) 98                                                                    ##STR99##                                                                      6.41 99                                                                        ##STR100##                                                                     CONSENSUS: 125                                                                 ##STR101##                                                                       Family IX                                                                    F13.46(4) 100                                                                  ##STR102##                                                                       Family X                                                                     F14.2 101                                                                      ##STR103##                                                                     F14.13(2) 102                                                                  ##STR104##                                                                       Family XI                                                                    6.72(2) 103                                                                    ##STR105##                                                                     6.42 104                                                                       ##STR106##                                                                       Family XII                                                                   6.30(5) 105                                                                    ##STR107##                                                                       Family XIII                                                                  6.52(2) 106                                                                    ##STR108##                                                                       Orphans                                                                      6.14 107                                                                       ##STR109##                                                                     6.36 108                                                                       ##STR110##                                                                     6.41 119                                                                       ##STR111##                                                                     6.44 110                                                                       ##STR112##                                                                     6.70 111                                                                       ##STR113##                                                                     6.76 112                                                                       ##STR114##                                                                     6.89 113                                                                       ##STR115##                                                                     6.81 114                                                                       ##STR116##                                                                     6.70 115                                                                       ##STR117##                                                                     F13.59 116                                                                     ##STR118##                                                                     F14.70 117                                                                     ##STR119##                                                               

                  TABLE IX                                                        ______________________________________                                        Dissociation Constants of Cloned Ligands to L-Selectin*                       Ligand  SEQ ID NO:     4° C.                                                                           Rm Temp                                       ______________________________________                                        Family I                                                                      F13.32  67             15.7   nM  14.9   nM                                   F13.48  74             15.9   nM  9.2    nM                                   F14.9   71             8.2    nM  15.4   nM                                   F14.21  72             2.3    nM  15.9   nM                                   F14.25  73             1300   nM                                              Family II                                                                     F14.12  78             5.8    pM  1.7    nM                                                    (0.68) (0.62)                                                                       16.2   nM  94     nM                                   F14.20  77             58     pM  1.0    nM                                                    (0.68) (0.28)                                                                       60     nM  48     nM                                   Family III                                                                    F13.57  89             3.0    nM  75     nM                                   Family V                                                                      F13.55  93             62     pM  1.5    nM                                   Family VI                                                                     F14.53  95             97     pM  142    nM                                                    (0.65)                                                                        14.5 nM                                                      Family VII                                                                    F13.42  96             2.0    nM  5.5    nM                                   F13.51  97             8.8    nM  18     nM                                   Family IX                                                                     F13.52             3.0    nM    25     nM                                     Family X                                                                      F14.2   101            1.8    nM  7.2    nM                                   F14.13  102            1.3    nM                                                               (0.74)                                                                        270  nM                                                      Orphans                                                                       F13.59  116            <5%      <5%                                           F14.70  117            2.0    nM  7.8    nM                                                    (0.75) (0.58)                                                                 254  nM    265      nM                                       ______________________________________                                         *Kds of monophasic binding ligands are indicated by a single number; the      high affinity K.sub.d (ie., K.sub.d1), the mole fraction binding with         K.sub.d1, and the low affinity K.sub.d (ie., K.sub.d2) are presented for      biphasic binding ligands.                                                

                  TABLE X                                                         ______________________________________                                        Specificity of Cloned Ligands to L-Selectin*                                  Ligand SEQ ID NO:                                                                              LS-Rg    ES-Rg  PS-Rg  CD22-Rg                               ______________________________________                                        Family I                                                                      F13.32 67        15.7   nM  <5%    17   μM                                                                             <5%                               F13.48 74        15.9   nM  <5%    720  nM  <5%                               F14.9  71        8.2    nM  <5%             <5%                               F14.21 72        2.3    nM  2.6  μM    <5%                                 F14.25 73        1300   nM                                                    Family II                                                                     F14.12 78        60     pM  47   nM  910  nM  <5%                             F14.20 77        58     pM  70   nM           <5%                                        (0.68)                                                                        60   nM                                                            Family III                                                                    F13.57 89        3.0    nM  2.7  μM    <5%                                 Family V                                                                      F13.55 93        62     pM  49   nM  5.8  μM                                                                             <5%                             Family VI                                                                     F14.53 95        97     pM  355  nM  5.2  μM                                                                             <5%                                        (0.65)                                                                        14.5 nM                                                            Family VII                                                                    F13.42 96        2.0    nM  4.4  μM    <5%                                 F13.51 97        8.8    nM  2.0  μM                                        Family IX                                                                     F13.52       3.0    nM    2.5  μM    <5%                                   Family X                                                                      F14.2  101       1.8    nM  1.9  μM                                                                             450  nM  <5%                             Orphans                                                                       F13.59 116       <5%      <5%           <5%                                   F14.70 117       2.0    nM  5.9  μM    <5%                                            (0.75)                                                                        254  nM                                                            ______________________________________                                         *Dissociation constants were determined at 4° C. in HSMC buffer.       When <5% binding was observed at the highest protein concentration, the K     is estimated to be >20 μM.                                            

                                      TABLE XI                                    __________________________________________________________________________    LS-Rg ssDNA SELEX                                                                     Total DNA                                                                           Total Prot.          % Eluted                                                                            % Eluted                                                                             Kd, nM  signal:bkgd           Round                                                                             Temp.                                                                             pmol  pmol DNA:Protein                                                                         Bead Vol.                                                                          Total Vol.                                                                         2 mM EDTA                                                                           50 mM EDTA                                                                           4 degrees                                                                             2                     __________________________________________________________________________                                                            mM                    Rnd 0                                           10,000                        Rnd 1                                                                              4  930   167  5.6   10 μL                                                                           ˜100 μL                                                                   n/a   5.5            50                    Rnd 2                                                                             25  400   167  2.4   10 μL                                                                           ˜100 μL                                                                   n/a   2.19           12                    Rnd 3                                                                             25  460   167  2.8   10 μL                                                                           ˜100 μL                                                                   n/a   2.55           25                    Rnd 4                                                                             25  100   16.7 6     10 μL                                                                           ˜100 μL                                                                   0.35  0.29           1.3                   Rnd 5                                                                             25  100   16.7 6     10 μL                                                                           ˜100 μL                                                                   0.23  0.08     967   3                     Rnd 6                                                                             25  1000  16.7 60    10 μL                                                                           ˜100 μL                                                                   1.42  0.38           4                     Rnd 7                                                                             25  100   16.7 6     10 μL                                                                           ˜100 μL                                                                   6.9   0.93      60   18                    Rnd 8                                                                             37  100   16.7 6     10 μL                                                                           ˜100 μL                                                                   1.9   0.31           9                     Rnd 9                                                                             25  10    1.67 6     10 μL                                                                           ˜100 μL                                                                   0.5   0.16   2.1     1.6                   Rnd 10                                                                            25  10    1.67 6     10 μL                                                                           ˜100 μL                                                                   2.2   0.57           5                     Rnd 11                                                                            25  2.5   0.42 6     10 μL                                                                           ˜100 μL                                                                   0.37  0.07    1.3 @ 25°                                                                     8.                    Rnd 12                                                                            25  2.5   0.42 6     10 μL                                                                           ˜100 μL                                                                   0.86  0.13           11                    Rnd 13                                                                            37  2.5   0.42 6     10 μL                                                                           ˜100 μL                                                                   0.7   0.35   0.44 @ 25°                                                                     5.                    Rnd 14                                                                            25  5     0.84 6     50 μL                                                                           ˜100 μL                                                                   2.8   0.76           4                     Rnd 15                                                                            25  1.25  0.21 6     50 μL                                                                           ˜100 μL                                                                   1.7   0.5    0.16 @ 25°                                                                     7.                    __________________________________________________________________________     Binding Buffer, Rounds 1-9                                                    10 mM HEPES, pH at room temp w/NaOH to 7.4                                    100 mM NaCl                                                                   1 mM MgCl.sub.2 -                                                             1 mM CaCl.sub.2 -                                                             5 mM KCl                                                                      Elution Buffers: replace divalent cations with EDTA                      

                                      TABLE XII                                   __________________________________________________________________________    L-Selectin ssDNA SELEX:                                                       Ligand Sequences                                                              Ligand                                                                              ID#                                                                     __________________________________________________________________________             Family 1                                                             204(3)                                                                              129                                                                               ##STR120##                                                          145   130                                                                               ##STR121##                                                          183(2)                                                                              131                                                                               ##STR122##                                                          230(2)                                                                              132                                                                               ##STR123##                                                          208(7)                                                                              133                                                                               ##STR124##                                                          227(5)                                                                              134                                                                               ##STR125##                                                          112   135                                                                               ##STR126##                                                          137   136                                                                               ##STR127##                                                          179(2)                                                                              137                                                                               ##STR128##                                                          182   138                                                                               ##STR129##                                                          190   139                                                                               ##STR130##                                                          193(2)                                                                              140                                                                               ##STR131##                                                          199   141                                                                               ##STR132##                                                          201(2)                                                                              142                                                                               ##STR133##                                                          203   143                                                                               ##STR134##                                                          207   144                                                                               ##STR135##                                                          216   145                                                                               ##STR136##                                                          233(5)                                                                              146                                                                               ##STR137##                                                          191   147                                                                               ##STR138##                                                          128(3)                                                                              148                                                                               ##STR139##                                                          111(2)                                                                              149                                                                               ##STR140##                                                          139   150                                                                               ##STR141##                                                          237   151                                                                               ##STR142##                                                          173   152                                                                               ##STR143##                                                          209   153                                                                               ##STR144##                                                          221   154                                                                               ##STR145##                                                          108   155                                                                               ##STR146##                                                          141   156                                                                               ##STR147##                                                          Truncates                                                                     201T1 172                                                                               ##STR148##                                                          201T3 173                                                                               ##STR149##                                                          Consensus:                                                                          168                                                                               ##STR150##                                                                   Family 2                                                             181(3)                                                                              157                                                                               ##STR151##                                                                   Family 4                                                             174(2)                                                                              158                                                                               ##STR152##                                                          122   159                                                                               ##STR153##                                                          239   160                                                                               ##STR154##                                                          Consensus:                                                                          169                                                                               ##STR155##                                                                   Family 5                                                             109   161                                                                               ##STR156##                                                          127   162                                                                               ##STR157##                                                          Consensus:                                                                          170                                                                               ##STR158##                                                                   Family 6                                                             196   163                                                                               ##STR159##                                                          229   164                                                                               ##STR160##                                                          Consensus:                                                                          171                                                                               ##STR161##                                                                   Family 7                                                             206(2)                                                                              165                                                                               ##STR162##                                                                   Orphans                                                              214   166                                                                               ##STR163##                                                          102   167                                                                               ##STR164##                                                          __________________________________________________________________________

                  TABLE XIII                                                      ______________________________________                                        Dissociation Constants of Cloned Ligands from ssDNA SELEX                     Against L-Selectin                                                                                   Room                                                   Ligand     SEQ ID NO:  Temperature                                                                              37° C.                               ______________________________________                                        Family 1                                                                      111        149         330     pM   11.8  nM                                  128        148         310     pM   1.8   nM                                  108        155         160     pM   8.5   nM                                  112        135         300     pM   23.2  nM                                  137        136         520     pM   0.65  nM                                  139        150         210     pM   6.8   nM                                  145        130         920     pM   8.8   nM                                  179        137         180     pM   590   pM                                  182        138         130     pM   2.0   nM                                  183        131         170     pM   1.0   nM                                  193        140         88      pM   970   pM                                  201        142         110     pM   1.2   nM                                  204        129         100     pM   3.7   nM                                  208        155         110     pM   380   pM                                  227        134         43      pM   160   pM                                  230        132         57      pM   260   pM                                  233        146         110     nM   380   pM                                  Family 2                                                                      181        157         84      pM   1.8   nM                                  Family 4                                                                      122        159         1.8     nM   2.1   nM                                  174        158         43      pM   370   pM                                  239        160         170     pM   1.6   nM                                  Family 5                                                                      109        161         190     pM   9.6   nM                                  127        162         1.0     nM   890   pM                                  Family 6                                                                      196        163         130     pM   3.4   nM                                  Family 7                                                                      206        165         330     pM   6.0   nM                                  Orphans                                                                       102        167         not determined                                                                           7.9   nM                                    214        166         660     pM   8.4   nM                                  Round 15 Pool          160     pM   660   pM                                  201T1*                              4.8   nM                                  201T3*                              43    nM                                  ______________________________________                                         *201T1 and 201T3 were made by solid state synthesis; the Kd of the            synthetic full length 201 control was 3.8 nM while that of enzymatically      synthecised 201 was 1.8 nM.                                              

                  TABLE XIV                                                       ______________________________________                                        Specificities of Cloned, ssDNA Ligands*                                       Ligand  SEQ ID NO: LS-Rg     ES-Rg   PS-Rg                                    ______________________________________                                        Family 1                                                                      111     149        1.1    nM   1.2  μM                                                                              840  nM                              201     142        110    nM   37   nM   1.0  μM                           204     129        450    pM   1.5  μM                                                                              2.9  μM                           227     134        64     pM   33   nM   560  nM                              230     132        44     pM   19   nM   600  nM                              233     146        120    pM   39   nM   420  nM                              Family 2                                                                      181     157        200    pM   37   nM   1.6  μM                           Family 4                                                                      122     159        340    pM   409  nM   420  nM                              174     158        46     pM   28   nM   380  nM                              Family 5                                                                      127     162        250    pM   1.3  μM                                                                              780  nM                              Family 6                                                                      196     163        220    pM   50   nM   3.4  μM                           Family 7                                                                      206     165        120    pM   100  nM   600  nM                              ______________________________________                                         *Kds were determined at room temperature. In assays with 700 nM CD22          β-Rg and 1.4 μM WGA less than 1% and 3% binding, respectively, wa     observed for all ligands suggesting that the dissociation constants are       greater than 100 μM for these proteins.                               

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 173                                                (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGGAAAAGCGAAUCAUACACAAGANNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNNNNNNNNNNNNNNNNNNNGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       UAAUACGACUCACUAUAGGGAAAAGCGAAUCAUACACAAGA41                                   (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       UUCUCGGUUGGUCUCUGGCGGAGC24                                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGGAAAAGCGAAUCAUACACAAGAAUGGUUGGCCUGGGCGCAGGCUUCGA50                          AGACUCGGCGGGAACGGGAAUGGCUCCGCCAGAGACCAACCGAGAA96                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGGAAAAGCGAAUCAUACACAAGACAGGCACUGAAAACUCGGCGGGAACG50                          AAAGUAGUGCCGACUCAGACGCGUGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGGAAAAGCGAAUCAUACACAAGAAGUCUGGCCAAAGACUCGGCGGGAAC50                          GUAAAACGGCCAGAAUUGCUCCGCCAGAGACCAACCGAGAA91                                   (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGGAAAAGCGAAUCAUACACAAGAGUAGGAGGUUCCAUCACCAGGACUCG50                          GCGGGAACGGAAGGUGAUGSGCUCCGCCAGAGACCAACCGAGAA94                                (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGGAAAAGCGAAUCAUACACAAGAACAAGGAUCGAUGGCGAGCCGGGGAG50                          GGCUCGGCGGGAACGAAAUCUGCUCCGCCAGAGACCAACCGAGAA95                               (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGGAAAAGCGAAUCAUACACAAGAUUGGGCAGGCAGAGCGAGACCGGGGG50                          CUCGGCGGGAACGGAACAGGAAUGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GGGAAAAGCGAAUCAUACACAAGAAAGGGAUGGGAUUGGGACGAGCGGCC50                          AAGACUCGGCGGGAACGAAGGGUGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGGAAAAGCGAAUCAUACACAAGACUCGGCGGGAACGAAAGUGUCAUGGU50                          AGCAAGUCCAAUGGUGGACUCUGCUCCGCCAGAGACCAACCGAGAA96                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGGAAAAGCGAAUCAUACACAAGACUCGGCGGGAACGUGAAGUGGGUAGG50                          UAGCUGAAGACGGUCUGGGCGCCAGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGGAAAAGCGAAUCAUACACAAGAAAGGGAUGGGAUUGGGACGAGCGGCC50                          AAGACUCGGCGGGAACGAAGGGUCCGCUCCGCCAGAGACCAACCGAGAA99                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGGAAAAGCGAAUCAUACACAAGACUCGGCGGGAACGAAGUGUGUGAGUA50                          ACGAUCACUUGGUACUAAAAGCCCGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GGGAAAAGCGAAUCAUACACAAGACUCGGCGGGAAUCGAAAGUGUACUGA50                          AUUAGAACGGUGGGCCUGCUCAUCGUGCUCCGCCAGAGACCAACCGAGAA100                         (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGGAAAAGCGAAUCAUACACAAGACUCGGCGGGAAUCGUAAUGUGGAUGA50                          UAGCACGAUGGCAGYAGUAGUCGGACCGCGCUCCGCCAGAGACCAACCGA100                         GAA103                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GGGAAAAGCGAAUCAUACACAAGACAGCGGCGGAGUCAGUGAAAGCGUGG50                          GGGGYGCGGGAGGUCUACCCUGACGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GGGAAAAGCGAAUCAUACACAAGACGGCUGUGUGUGGUAGCGUCAUAGUA50                          GGAGUCGUCACGAACCAAGGCGCUCCGCCAGAGACCAACCGAGAA95                               (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GGGAAAAGCGAAUCAUACACAAGACGGCUGUGUGGUGUUGGAGCGUCAUA50                          GUAGGAGUCGUCACGAACCAAGGCGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GGGAAAAGCGAAUCAUACACAAGACGAUGCGAGGCAAGAAAUGGAGUCGU50                          UACGAACCCUCUUGCAGUGCGCGGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GGGAAAAGCGAAUCAUACACAAGACGUGCGGAGCAAAUAGGGGAUCAUGG50                          AGUCGUACGAACCGUUAUCGCGCUCCGCCAGAGACCAACCGAGAA95                               (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GGGAAAAGCGAAUCAUACACAAGACUGGGGAGCAGGAUAUGAGAUGUGCG50                          GGGCAAUGGAGUCGUGACGAACCGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GGGAAAAGCGAAUCAUACACAAGAGUCCGCCCCCAGGGAUGCAACGGGGU50                          GGCUCUAAAAGGCUUGGCUAAGCUCCGCCAGAGACCAACCGAGAA95                               (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GGGAAAAGCGAAUCAUACACAAGAGAGAAUGAGCAUGGCCGGGGCAGGAA50                          GUGGGUGGCAACGGAGGCCAGCUCCGCCAGAGACCAACCGAGAA94                                (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGGAAAAGCGAAUCAUACACAAGAGAUACAGCGCGGGUCUAAAGACCUUG50                          CCCCUAGGAUGCAACGGGGUGGCUCCGCCAGAGACCAACCGAGAA95                               (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GGGAAAAGCGAAUCAUACACAAGAUGAAGGGUGGUAAGAGAGAGUCUGAG50                          CUCGUCCUAGGGAUGCAACGGCAGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GGGAAAAGCGAAUCAUACACAAGACAAACCUGCAGUCGCGCGGUGAAACC50                          UAGGGUUGCAACGGUACAUCGCUGUGCUCCGCCAGAGACCAACCGAGAA99                           (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GGGAAAAGCGAAUCAUACACAAGAGUGGACUGGAAUCUUCGAGGACAGGA50                          ACGUUCCUAGGGAUGCAACGGACGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GGGAAAAGCGAAUCAUACACAAGAGUGUACCAAUGGAGGCAAUGCUGCGG50                          GAAUGGAGGCCUAGGGAUGCAACGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GGGAAAAGCGAAUCAUACACAAGAGUCCCUAGGGAUGCAACGGGCAGCAU50                          UCGCAUAGGAGUAAUCGGAGGUCGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GGGAAAAGCGAAUCAUACACAAGAGCCUAGGGAUGCAACGGCGAAUGGAU50                          AGCGAUGUCGUGGACAGCCAGGUGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GGGAAAAGCGAAUCAUACACAAGAAUCGAACCUAGGGAUGCAACGGUGAA50                          GGUUGUGAGGAUUCGCCAUUAGGCGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GGGAAAAGCGAAUCAUACACAAGAGCUAGGGAUGCCGCAGAAUGGUCGCG50                          GAUGUAAUAGGUGAAGAUUGUUGCGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GGGAAAAGCGAAUCAUACACAAGAGGACCUAGGGAUGCAACGGUCCGACC50                          UUGAUGCGCGGGUGUCCAAGCUACGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GGGAAAAGCGAAUCAUACACAAGAAAGGGAGGAGCUAGAGAGGGAAAGGU50                          UACUACGCGCCAGAAUAGGAUGUGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GGGAAAAGCGAAUCAUACACAAGACCAACGUACAUCGCGAGCUGGUGGAG50                          AGUUCAUGAGGGUGUUACGGGGUGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GGGAAAAGCGAAUCAUACACAAGACCCAACGUGUCAUCGCGAGCUGGCGG50                          AGAGUUCAUGAGGGUUACGGGUGCUCCGCCAGAGACCAACCGAGAA96                              (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      GGGAAAAGCGAAUCAUACACAAGAGUUGGUGCGAGCUGGGGCGGCGAGAA50                          GGUAGGCGGUCCGAGUGUUCGAAUGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GGGAAAAGCGAAUCAUACACAAGACUGGCAAGRAGUGCGUGAGGGUACGU50                          UAGGGGUGUUUGGGCCGAUCGCAUGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GGGAAAAGCGAAUCAUACACAAGAUUGGUCGUACUGGACAGAGCCGUGGU50                          AGAGGGAUUGGGACAAAGUGUCAGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GGGAAAAGCGAAUCAUACACAAGAUGUGAGAAAGUGGCCAACUUUAGGAC50                          GUCGGUGGACUGYGCGGGUAGGCUCGCUCCGCCAGAGACCAACCGAGAA99                           (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GGGAAAAGCGAAUCAUACACAAGACAGGCAGAUGUGUCUGAGUUCGUCGG50                          AGUAGACGUCGGUGGACGCGGAACGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GGGAAAAGCGAAUCAUACACAAGAUGUGAUUAGGCAGUUGCAGCCGCCGU50                          GCGGAGACGUGACUCGAGGAUUCGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GGGAAAAGCGAAUCAUACACAAGAUGCCGGUGGAAAGGCGGGUAGGUGAC50                          CCGAGGAUUCCUACCAAGCCAUGCUCCGCCAGAGACCAACCGAGAA96                              (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GGGAAAAGCGAAUCAUACACAAGAGAGGUGRAUGGGAGAGUGGAGCCCGG50                          GUGACUCGAGGAUUCCCGUGCUCCGCCAGAGACCAACCGAGAA93                                 (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GGGAAAAGCGAAUCAUACACAAGAGUCAUGCUGUGGCUGAACAUACUGGU50                          GAAAGUUCAGUAGGGUGGAUACAGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GGGAAAAGCGAAUCAUACACAAGACCGGGGAUGGUGAGUCGGGCAGUGUG50                          ACCGAACUGGUGCCCGCUGAGAGCUCCGCCAGAGACCAACCGAGAA96                              (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GGGAAAAGCGAAUCAUACACAAGAACACUAACCAGGUCUCUGAACGCGGG50                          ACGGAGGUGUGGGCGAGGUGGAAGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GGGAAAAGCGAAUCAUACACAAGACCGUCUCCCGAGAACCAGGCAGAGGA50                          CGUGCUGAAGGAGCUGCAUCUAGAAGCUCCGCCAGAGACCAACCGAGAA99                           (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GGGAAAAGCGAAUCAUACACAAGACCGUCUCCGAGAACCAGGCAGAGGAG50                          GUGCUGAAGGRGCUGGCAUCUACAAGCUCCGCCAGAGACCAACCGAGAA99                           (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GGGAAAAGCGAAUCAUACACAAGACCCGCACAUAAUGUAGGGAACAAUGU50                          UAUGGCGGAAUUGAUAACCGGUGCUCCGCCAGAGACCAACCGAGAA96                              (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GGGAAAAGCGAAUCAUACACAAGACGAUGUUAGCGCCUCCGGGAGAGGUU50                          AGGGUCGUGCGGNAAGAGUGAGGUGCUCCGCCAGAGACCAACCGAGAA98                            (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      GGGAAAAGCGAAUCAUACACAAGAGGUACGGGCGAGACGAGAUGGACUUA50                          UAGGUCGAUGAACGGGUAGCAGCUCGCUCCGCCAGAGACCAACCGAGAA99                           (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GGGAAAAGCGAAUCAUACACAAGACGGUUGCUGAACAGAACGUGAGUCUU50                          GGUGAGUCGCACAGAUUGUCCUGCUCCGCCAGAGACCAACCGAGAA96                              (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GGGAAAAGCGAAUCAUACACAAGAACUGAGUAAGGUCUGGCGUGGCAUUA50                          GGUUAGUGGGAGGCUUGGAGUAGGCUCCGCCAGAGACCAACCGAGAA97                             (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      AAGACUCGGCGGGAACGAAA20                                                        (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GGAGUCGUGACGAACC16                                                            (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CCUAGGGAUGCAACGG16                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      RCUGGGAGRGUGGGUGUU18                                                          (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      UGUGNNNNAGUNNNNNNNNNUAGACGUCGGUGGACNNNGCGG42                                  (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GGGNNNGUGACYCGRGGAYUC21                                                       (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      UGANCNNACUGGUGNNNGNGNAG23                                                     (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      GUCUCYGAACNNGGNAGGANGUGNUGGAGNUG32                                            (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GGGAGGACGAUGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      TAATACGACTCACTATAGGGAGGACGATGCGG32                                            (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      TCGGGCGAGTCGTCCTG17                                                           (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      GGGAGGACGAUGCGGCGCGUAUGUGUGAAAGCGUGUGCACGGAGGCGUCU50                          ACAAUCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      GGGAGGACGAUGCGGGGCAUUGUGUGAAUAGCUGAUCCCACAGGUAACAA50                          CAGCACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      GGGAGGACGAUGCGGUAAUGUGUGAAUCAAGCAGUCUGAAUAGAUUAGAC50                          AAAAUCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      GGGAGGACGAUGCGGAUGUGUGAGUAGCUGAGCGCCCGAGUAUGAWACCU50                          GACUACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      GGGAGGACGAUGCGGAAACCUUGAUGUGUGAUAGAGCAUCCCCCAGGCGA50                          CGUACCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      GGGAGGACGAUGCGGUUGAGAUGUGUGAGUACAAGCUCAAAAUCCCGUUG50                          GAGGCAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      GGGAGGACGAUGCGGUAGAGGUAGUAUGUGUGGGAGAUGAAAAUACUGUG50                          GAAAGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      GGGAGGACGAUGCGGAAAGUUAUGAGUCCGUAUAUCAAGGUCGACAUGUG50                          UGAAUCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      GGGAGGACGAUGCGGCACGAAAAACCCGAAUUGGGUCGCCCAUAAGGAUG50                          UGUGACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      GGGAGGACGAUGCGGGUAAAGAGAUCCUAAUGGCUCGCUAGAUGUGAUGU50                          GAAACCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      GGGAGGACGAUGCGGUAACAACAAUCAAGGCGGGUUCACCGCCCCAGUAU50                          GAGUGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      GGGAGGACGAUGCGGUAACAACAAUCAAGGCGGGUUYACCGCCCCAGUAU50                          GAGUACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      GGGAGGACGAUGCGGUAACAACAAUCAAGGCGGGUUYACCGCUCCAGUAU50                          GAGUACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      GGGAGGACGAUGCGGUAACAACAAUCAAGGCGGGUUCACCGCCCCAGUAU50                          GAGUGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      GGGAGGACGAUGCGGACCAAGCAAUCUAUGGUCGAACGCUACACAUGAAU50                          GACGUCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      GGGAGGACGAUGCGGGAACAUGAAGUAAUCAAAGUCGUACCAAUAUACAG50                          GAAGCCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      GGGAGGACGAUGCGGGACAUGAAGUAAGACCGUCACAAUUCGAAUGAUUG50                          AAUACAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      GGGAGGACGAUGCGGGAACAUGAAGUAAAAAGUCGACGAAUUAGCUGUAA50                          CCAAAACAGACGACUCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      GGGAGGACGAUGCGGGAACAUGAAGUAAAAGUCUGAGUUAGUAAAUUACA50                          GUGAUCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      GGGAGGACGAUGCGGGAACUUGAAGUUGAANUCGCUAAGGUUAUGGAUUC50                          AAGAUUCAGACGACUCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      GGGAGGACGAUGCGGAACAUGAAGUAAUAAGUCGACGUAAUUAGCUGUAA50                          CUAAACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      GGGAGGACGAUGCGGAACAUGAAGUAAAAGUCUGAGUUAGAAAUUACAAG50                          UGAUCAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      GGGAGGACGAUGCGGUAACAUAAAGUAGCGCGUCUGUGAGAGGAAGUGCC50                          UGGAUCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      GGGAGGACGAUGCGGAUAGAACCGCAAGGAUAACCUCGACCGUGGUCAAC50                          UGAGACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      GGGAGGACGAUGCGGUAAGAACCGCUAGCGCACGAUCAAACAAAGAGAAA50                          CAAACAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      GGGAGGACGAUGCGGUUCUCUCCAAGAACYGAGCGAAUAAACSACCGGAS50                          UCACACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      GGGAGGACGAUGCGGUGUCUCUCCUGACUUUUAUUCUUAGUUCGAGCUGU50                          CCUGGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      GGGAGGACGAUGCGGCCGUACAUGGUAARCCUCGAAGGAUUCCCGGGAUG50                          AUCCCCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      GGGAGGACGAUGCGGUCCCAGAGUCCCGUGAUGCGAAGAAUCCAUUAGUA50                          CCAGACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      GGGAGGACGAUGCGGGAUGUAAAUGACAAAUGAACCUCGAAAGAUUGCAC50                          ACUCCAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      GGGAGGACGAUGCGGAUGUAAAUCUAGGCAGAAACGUAGGGCAUCCACCG50                          CAACGACAGACGACUCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      GGGAGGACGAUGCGGAUAACCCAAGCAGCNUCGAGAAAGAGCUCCAUAGA50                          UGAUCAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:99:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                      GGGAGGACGAUGCGGCAAAGCACGCGUAUGGCAUGAAACUGGCANCCCAA50                          GUAAGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:100:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                                     GGGAGGACGAUGCGGCAAAAGGUUGACGUAGCGAAGCUCUCAAAAUGGUC50                          AUGACCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:101:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                                     GGGAGGACGAUGCGGAAGUGAAGCUAAAGCGGAGGGCCAUUCAGUUUCNC50                          ACCACAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:102:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                                     GGGAGGACGAUGCGGAAGUGAAGCUAAAGSGGAGGGCCACUCAGAAACGC50                          ACCACAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:103:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                                     GGGAGGACGAUGCGGCACCGCUAAGCAGUGGCAUAGCCCAGUAACCUGUA50                          AGAGACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:104:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                                     GGGAGGACGAUGCGGCACGCUAAGCAGUGGCAUAGCGWAACCUGUAAGAG50                          ACAGACGACUCGCCCGA67                                                           (2) INFORMATION FOR SEQ ID NO:105:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                                     GGGAGGACGAUGCGGAGAUUACCAUAACCGCGUAGUCGAAGACAUAUAGU50                          AGCGACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:106:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                                     GGGAGGACGAUGCGGACUCGGGUAGAACGCGACUUGCCACCACUCCCAUA50                          AAGACCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:107:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                                     GGGAGGACGAUGCGGUCAGAACUCUGCCGCUGUAGACAAAGAGGAGCUUA50                          GCGAACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:108:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                                     GGGAGGACGAUGCGGAAUGAGCAUCGAGAGAGCGCGAACUCAUCGAGCGU50                          ACUAACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:109:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                                     GGGAGGACGAUGCGGCAAAGCACGCGUAUGGCAUGAAACUGGCANCCCAA50                          GUAAGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:110:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                                     GGGAGGACGAUGCGGGAUGCAGCAACCUGAAAACGGCGUCCACAGGUAAU50                          AACAGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:111:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                                     GGGAGGACGAUGCGGAAACUCGCUACAAACACCCAAUCCUAGAACGUUAU50                          GGAGACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:112:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                                     GGGAGGACGAUGCGGCUAGCAUAGCCACCGGAACAGACAGAUACGAGCAC50                          GAUCACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:113:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                                     GGGAGGACGAUGCGGGAUUCGGAGUACUGAAAAACAACCCUCAAAAGUGC50                          AUAGGCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:114:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                                     GGGAGGACGAUGCGGGUCCAGGACGGACCGCAGCUGUGAUACAAUCGACU50                          UACACCAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:115:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                                     GGGAGGACGAUGCGGAAACUCGCUACAAACACCCAAUCCUAGAACGUUAU50                          GGAGACAGACGACUCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:116:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                                     GGGAGGACGAUGCGGCGGCCCUUAUCGGAGGUCUGCGCCACUAAUUACAU50                          CCACCAGACGACUCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO:117:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                                     GGGAGGACGAUGCGGUCCAGAGCGUGAAGAUCAACGUCCCGGNGUCGAAG50                          ACAGACGACUCGCCCGA67                                                           (2) INFORMATION FOR SEQ ID NO:118:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                                     AUGUGUGA8                                                                     (2) INFORMATION FOR SEQ ID NO:119:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                                     CAACAAUCAUGAGUR15                                                             (2) INFORMATION FOR SEQ ID NO:120:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                                     AACAUGAAGUAAGUCARUUAG21                                                       (2) INFORMATION FOR SEQ ID NO:121:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:                                     AGAACCGCWAG11                                                                 (2) INFORMATION FOR SEQ ID NO:122:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:                                     UCUCUCC7                                                                      (2) INFORMATION FOR SEQ ID NO:123:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:                                     CGAAGAAUYC10                                                                  (2) INFORMATION FOR SEQ ID NO:124:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:                                     AUGUAAAU8                                                                     (2) INFORMATION FOR SEQ ID NO:125:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine                            (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:                                     AACCCAAG8                                                                     (2) INFORMATION FOR SEQ ID NO:126:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:                                     CTACCTACGATCTGACTAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNNNNNGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:127:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:                                     CTACCTACGATCTGACTAGC20                                                        (2) INFORMATION FOR SEQ ID NO:128:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:                                     AGGAACTACATGAGAGTAAGC21                                                       (2) INFORMATION FOR SEQ ID NO:129:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:                                     CTACCTACGATCTGACTAGCGGAACACGTGAGGTTTACAAGGCACTCGAC50                          GTAAACACTTGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:130:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:                                     CTACCTACGATCTGACTAGCCCCCGAAGAACATTTTACAAGGTGCTAAAC50                          GTAAAATCAGGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:131:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:                                     CTACCTACGATCTGACTAGCGGCATCCCTGAGTCATTACAAGGTTCTTAA50                          CGTAATGTACGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:132:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:                                     CTACCTACGATCTGACTAGCTGCACACCTGAGGGTTACAAGGCGCTAGAC50                          GTAACCTCTCGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:133:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:                                     CTACCTACGATCTGACTAGCCACGTTTCAAGGGGTTACACGAAACGATTC50                          ACTCCTTGGCGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:134:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:                                     CTACCTACGATCTGACTAGCCGGACATGAGCGTTACAAGGTGCTAAACGT50                          AACGTACTTGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:135:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:                                     CTACCTACGATCTGACTAGCCGCATCCACATAGTTCAAGGGGCTACACGA50                          AATATTGCAGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:136:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:                                     CTACCTACGATCTGACTAGCTACCCCTTGGGCCTCATAGACAAGGTCTTA50                          AACGTTAGCGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:137:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:                                     CTACCTACGATCTGACTAGCCACATGCCTGACGCGGTACAAGGCCTGGAC50                          GTAACGTTGGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:138:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:                                     CTACCTACGATCTGACTAGCTAGTGCTCCACGTATTCAAGGTGCTAAACG50                          AAGACGGCCTGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:139:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:                                     CTACCTACGATCTGACTAGCAGCGATGCAAGGGGCTACACGCAACGATTT50                          AGATGCTCTGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:140:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:                                     CTACCTACGATCTGACTAGCCCAGGAGCACAGTACAAGGTGTTAAACGTA50                          ATGTCTGGTGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:141:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:                                     CTACCTACGATCTGACTAGCACCACACCTGGGCGGTACAAGGAGTTATCC50                          GTAACGTGTGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:142:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:                                     CTACCTACGATCTGACTAGCCAAGGTAACCAGTACAAGGTGCTAAACGTA50                          ATGGCTTCGGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:143:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:                                     CTACCTACGATCTGACTAGCACCCCCGACCCGAGTACAAGGCATTCGACG50                          TAATCTGGTGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:144:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:                                     CTACCTACGATCTGACTAGCCAGTACAAGGTGTTAAACGTAATGCCGATC50                          GAGTTGTATGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:145:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:                                     CTACCTACGATCTGACTAGCACAACGAGTACAAGGAGATAGACGTAATCG50                          GCGCAGGTATCGCTTACTCTCATGTAGTTCC81                                             (2) INFORMATION FOR SEQ ID NO:146:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:                                     CTACCTACGATCTGACTAGCCACGACAGAGAACAAGGCGTTAGACGTTAT50                          CCGACCACGGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:147:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:                                     CTACCTACGATCTGACTAGCAGGGAGAACAAGGTGCTAAACGTTTATCTA50                          CACTTCACCTGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:148:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:                                     CTACCTACGATCTGACTAGCAGGACCAAGGTGTTAAACGGCTCCCCTGGC50                          TATGCCTCTTGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:149:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:                                     CTACCTACGATCTGACTAGCTACACAAGGTGCTAAACGTAGAGCCAGATC50                          GGATCTGAGCGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:150:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:                                     CTACCTACGATCTGACTAGCGGACAAGGCACTCGACGTAGTTTATAACTC50                          CCTCCGGGCCGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:151:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:                                     CTACCTACGATCTGACTAGCTACACAAGGGGCCAAACGGAGAGCCAGACG50                          CGGATCTGACAGCTTACTCTCATGTAGTTCC81                                             (2) INFORMATION FOR SEQ ID NO:152:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:                                     CTACCTACGATCTGACTAGCCGGCTATACNNGGTGCTAAACGCAGAGACT50                          CGATCAACAGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:153:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:                                     CTACCTACGATCTGACTAGCGAGTAGCCAAGGCGTTAGACGGAGGGGGAA50                          TGGAAGCTTGGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:154:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:                                     CTACCTACGATCTGACTAGCGAGTAGCCAAGGCGTTAGACGGAGGGGGAA50                          TGGGCTTACTCTCATGTAGTTCC73                                                     (2) INFORMATION FOR SEQ ID NO:155:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:                                     CTACCTACGATCTGACTAGCGAGTAGCCAAGGCGTTAGACGGAGGGGGAA50                          TGTGAGCACAGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:156:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:                                     CTACCTACGATCTGACTAGCTAGCTCCACACACAASSCGCRGCACATAGG50                          GGATATCTGGGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:157:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:                                     CTACCTACGATCTGACTAGCCATCAAGGACTTTGCCCGAAACCCTAGGTT50                          CACGTGTGGGGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:158:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:                                     CTACCTACGATCTGACTAGCCATTCACCATGGCCCCTTCCTACGTATGTT50                          CTGCGGGTGGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:159:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:                                     CTACCTACGATCTGACTAGCGCAACGTGGCCCCGTTTAGCTCATTTGACC50                          GTTCCATCCGGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:160:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:                                     CTACCTACGATCTGACTAGCCCACAGACAATCGCAGTCCCCGTGTAGCTC50                          TGGGTGTCTGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:161:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:                                     CTACCTACGATCTGACTAGCCCACCGTGATGCACGATACATGAGGGTGTG50                          TCAGCGCATGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:162:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:                                     CTACCTACGATCTGACTAGCCGAGGTAGTCGTTATAGGGTRCRCACGACA50                          CAAARCRGTRGCTTACTCTCATGTAGTTCC80                                              (2) INFORMATION FOR SEQ ID NO:163:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:                                     CTACCTACGATCTGACTAGCTGGCGGTACGGGCCGTGCACCCACTTACCT50                          GGGAAGTGAGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:164:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:                                     CTACCTACGATCTGACTAGCCTCTGCTTACCTCATGTAGTTCCAAGCTTG50                          GCGTAATCATGGCTTACTCTCATGTAGTTCC81                                             (2) INFORMATION FOR SEQ ID NO:165:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:                                     CTACCTACGATCTGACTAGCAGCGTTGTACGGGGTTACACACAACGATTT50                          AGATGCTCTGCTTACTCTCATGTAGTTCC79                                               (2) INFORMATION FOR SEQ ID NO:166:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:                                     CTACCTACGATCTGACTAGCTGATGCGACTTTAGTCGAACGTTACTGGGG50                          CTCAGAGGACAGCTTACTCTCATGTAGTTCC81                                             (2) INFORMATION FOR SEQ ID NO:167:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:                                     CTACCTACGATCTGACTAGCCGAGGATCTGATACTTATTGAACATAMCCG50                          CACNCAGGCTTGCTTACTCTCATGTAGTTCC81                                             (2) INFORMATION FOR SEQ ID NO:168:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:                                     TACAAGGYGYTAVACGTA18                                                          (2) INFORMATION FOR SEQ ID NO:169:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:                                     GGCCCCGT8                                                                     (2) INFORMATION FOR SEQ ID NO:170:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:                                     RCACGAYACA10                                                                  (2) INFORMATION FOR SEQ ID NO:171:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:                                     CTTACCT7                                                                      (2) INFORMATION FOR SEQ ID NO:172:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:                                     CAAGGTAACCAGTACAAGGTGCTAAACGTAATGGCTTCG39                                     (2) INFORMATION FOR SEQ ID NO:173:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:                                     GTAACCAGTACAAGGTGCTAAACGTAATGGCTTCG35                                         __________________________________________________________________________

We claim:
 1. A method for identifying nucleic acid ligands and nucleicacid ligand sequences to a lectin comprising:a) contacting a candidatemixture of nucleic acids with a lectin, wherein nucleic acids having anincreased affinity to said lectin relative to the candidate mixture maybe partitioned from the remainder of the candidate mixture; b)partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; and c) amplifying the increased affinity nucleicacids to yield a mixture of nucleic acids enriched for nucleic acidsequences with relatively higher affinity and specificity for binding tosaid lectin, whereby nucleic acid ligands to said lectin may beidentified.
 2. The method of claim 1 further comprising:d) repeatingsteps a), b) and c).
 3. The method of claim 1 wherein said candidatemixture is comprised of single-stranded nucleic acids.
 4. The method ofclaim 3 wherein said single-stranded nucleic acids are ribonucleicacids.
 5. The method of claim 4 wherein said nucleic acids comprisemodified ribonucleic acids.
 6. The method of claim 5 wherein saidnucleic acids comprise 2'-amino (2'-NH₂) modifed ribonucleic acids. 7.The method of claim 3 wherein said single-stranded nucleic acids aredeoxyribonucleic acids.
 8. The method of claim 1 wherein said lectin isselected from the group consisting of a mammalian lectin, a plantlectin, a microbial lectin and a viral lectin.
 9. The method of claim 1wherein said lectin is wheat germ agglutinin.
 10. The method of claim 1wherein said lectin is serum mannose binding protein.